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 && Declaration  I, Felix Paul Anton Mayer, declare that the current thesis entitled “Unravelling the mechanism of action of new psychoactive substances and their phase 1 metabolites“ was conducted under supervision of Prof. Dr. Harald H. Sitte at the Institute of Pharmacology, Medical University of Vienna. Except for the manuscripts embedded herein, the thesis at hand has not been submitted or published elsewhere. Further, I declare that this thesis was written in accordance with the guidelines for good scientific practice of the Medical University of Vienna.

With respect to the publication embedded, entitled “Phase I metabolites of display biological activity as substrates at monoamine transporters”, I declare that I had the original idea for the study, i.e. testing the phase 1 metabolites of mephedrone for their bioactive properties. Further, I performed and analysed uptake inhibition and release experiments in HEK293 cells. Together with Prof. Dr. Harald H. Sitte and Dr. Michael H. Baumann, I planned the design of the study and wrote the initial version of the manuscript. Dr. Laurin Wimmer synthesized the metabolites in the laboratory of Prof. Dr. Marko D. Mihovilovic. The release assays in rat brain synaptosomes, microdialysis and behavioural pharmacology studies were planned, designed and conducted in the laboratory of Dr. Michael H. Baumann.

With respect to the publication embedded, entitled “Pharmacological characterization of fluorinated “research chemicals””, I declare that I performed and analysed uptake inhibition and release studies in HEK293 cells. Dr. Simon D. Brandt provided the fluorinated phenmetrazine research chemicals. Release studies in rat brain synaptosomes were performed in the laboratories of Dr. Michael H. Baumann and Dr. Bruce E. Blough. Together with Prof. Harald H. Sitte, I planned the design of the study. I wrote the first draft of the manuscript, which was corrected and approved by Prof. Dr. Harald H. Sitte. We received significant input from all other co-authors, especially Dr. Michael H. Baumann.

With respect to the book chapter embedded in this thesis, entitled “Application of a combined approach to identify new psychoactive drugs and decipher their mechanisms at monoamine transporters”, I declare that I wrote the first draft of the book chapter, which was then corrected and approved by Prof. Dr. Harald H. Sitte.  &&& We received significant input from all other authors, especially Dr. Rainer Schmid and the editors Dr. Michael H. Baumann and Prof. Dr. Richard A. Glennon.

The work embedded in this thesis was supported by a DOC-fellowship of the Austrian Academy of Sciences and the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung, FWF grant W1232 DK "Molecular Drug Targets" and F3506 SFB35 "Transmembrane Transporters in Health and Disease").

Vienna, January 2017

Felix P.A. Mayer 

&3 Abstract  Psychostimulant abuse constitutes a growing problem on a global scale, with no effective treatments for psychostimulant being available at present. In addition to well-characterized and regulated , such as or 3,4- methylenedioxymethamphetamine (MDMA, “ecstasy”), the drug markets are flooded with new psychoactive substances (NPS). NPS are often referred to as “bath salts” or “research chemicals” and are sold as legal alternatives to scheduled substances. Due to chemical modifications, NPS bypass regulations and result in an overwhelming variety of substances with unknown pharmacology. The rewarding, stimulating and addictive properties of psychostimulants arise from their ability to elevate the extracellular concentrations of the monoamines , and . This is achieved by disrupting the function of the transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Under physiological conditions, DAT, NET and SERT mediate the of exocytically-released monoamines and tightly regulate the strength of monoaminergic signaling. Despite similarities in their ability to enhance monoaminergic transmission, psychostimulants differ in their specific mechanisms of action. Cocaine-like drugs act as non-transported inhibitors, whereas - like releasers, including some types of NPS, cause efflux of monoamines through the transporters. Furthermore, releasers act as substrates of DAT, NET and SERT and gain access to the . In the cytosol, releasers may disrupt the vesicular storage pools of monoamines and can exert neurotoxic effects. Thus, understanding how NPS act on DAT, NET and SERT is crucial to establishing the mechanism of action of this new class of compounds. During my thesis, I tested whether the phase 1 metabolites of 4-methylmethcathinone (mephedrone) possess psychoactive properties similar to the phase 1 metabolites of MDMA, and thus might contribute to the effects of mephedrone. Mephedrone became famous as part of the group of compounds called “bath salts” and is still abused as an alternative to MDMA. Results obtained from radiotracer-flux experiments showed that the phase 1 metabolites of mephedrone act as releasers at DAT, NET and SERT. In vivo studies in rats identified one metabolite that elevated extracellular dopamine and serotonin in the and triggered locomotion upon systemic administration. Future studies shall investigate the of mephedrone and its metabolites in brain and plasma to estimate the overall contribution of the metabolites to the effects  3 of mephedrone. The second study embedded in this thesis provides a pharmacological characterization of the NPS 3-fluorophenmetrazine (3-FPM) and its positional 2-FPM and 4-FPM. The chemical structure of 3-FPM is based on the scheduled drug phenmetrazine. 2-, 3- and 4-FPM were identified as releasers at DAT, NET and SERT. The marked affinity of each FPM at DAT and NET suggests addictive properties and enhanced likelihood for abuse. In conclusion, the projects embedded in this thesis reveal that the metabolites of mephedrone might contribute to the overall-effects of mephedrone in vivo and provide a pharmacological characterization of potential future drugs of abuse. Further, the techniques applied herein may serve as a guideline for unraveling the mode of action of psychostimulants at monoamine transporters.

3& Zusammenfassung  Missbrauch von Psychostimulanzien stellt ein globales und im Wachsen begriffenes Problem dar. Die gleichzeitige Abwesenheit von effektiven Behandlungsstrategien, um die Abhängigkeit von diesen Substanzen zu behandeln, verleiht diesem Problem zusätzliche Brisanz. Neben den etablierten und intensiv beforschten Psychostimulanzien, wie etwa Cocain oder 3,4-Methylenedioxymethylamphetamin (MDMA; „ecstasy“), drängen neue psychoaktive Substanzen (NPS) auf die globalen Drogenmärkte. Diese auch als „Badesalze“ und „Research Chemicals“ bezeichneten Substanzen werden als legale Alternativen zu den regulierten und illegalen Stimulanzien konsumiert. Aufgrund von chemischen Modifikationen unterwandern NPS die Gesetzgebung und bringen eine Vielfalt an Substanzen hervor, deren pharmakologisches und toxikologisches Profil gänzlich unbekannt ist. Die stimulierende und belohnende Wirkung von Psychostimulanzien basiert auf deren Fähigkeit, die extrazelluläre Konzentration der Monoamine Dopamin, Serotonin und Noradrenalin zu erhöhen. Unter physiologischen Bedingungen bewerkstelligen hochaffine Transportproteine die Wiederaufnahme dieser und terminieren somit deren Wirkung an pre- und postsynaptischen Rezeptoren. Die Transporter für Dopamin (DAT), Noradrenalin (NET) und Serotonin (SERT) stellen den Angriffspunkt für Psychostimulanzien dar, wobei diese unterschiedliche Wirkungen an den Transportproteinen entfalten: Cocain-artige Substanzen agieren als nicht-transportierbare Inhibitoren und hemmen somit die Wiederaufnahme von exozytotisch freigesetzten Monoaminen. Amphetamin-artige Stimulanzien kehren die Transportrichtung von DAT, NET und SERT um, wodurch eine nicht-exozytotische Freisetzung von Monoaminen ausgelöst wird. Zusätzlich gelangen Amphetamin- artige Substanzen als Substrate von DAT, NET und SERT in das Zytosol und können somit in einigen Fällen die vesikulären Monoaminspeicher depletieren, womit potentielle neurotoxische Effekte assoziiert sind. Daraus resultierend ergibt sich die Notwendigkeit, die Wirkung von stimulierend wirkenden NPS an DAT, NET und SERT zu entschlüsseln. Während meiner Dissertation wurde der Frage nachgegangen, ob -analog der Phase 1 Metaboliten von MDMA- die Phase I Metaboliten von 4-Methylmethcathinon (Mephedron) psychoaktive Eigenschaften besitzen und somit zu den subjektiven Effekten von Mephedron betragen könnten. Mephedron erlangte als „Badesalz“ Berühmtheit und wird Gegenwärtig als MDMA- Alternative konsumiert. Experimente mit radioaktiv markierten Substraten von 3&& Monoamintransportern konnten zeigen, dass die Phase 1 Metaboliten von Mephedron mit DAT, NET und SERT interagieren und als Amphetamin-artige Substanzen eine nicht-exozytotische Freisetzung von Monoaminen bewirken. Mittels in vivo Studien in Ratten konnte ein Metabolit identifiziert werden, welcher nach systemischer Administration Dopamin und Serotonin im zentralen Nervensystem freisetzt und eine Steigerung der basalen Bewegungsaktivität auslöst. Weitere Studien sind geplant, um die Pharmakokinetik von Mephedron und den zugehörigen Metaboliten im zentralen Nervensystem und Blutplasma zu entschlüsseln, sodass die Kontribution der Metaboliten zu den zentralen Effekten von Mephedron abgeschätzt werden kann. Die zweite Studie, welche in diese Dissertation eingebettet wurde, enthält eine pharmakologische Charakterisierung der NPS 3-Fluorophenmetrazin (3- FPM) und der Stellungsisomere 2- und 4-FPM, welche Abkömmlinge der psychoaktiven Substanz Phenmetrazin darstellen. 2-, 3- und 4-FPM konnten als Amphetamin-artige Substanz - und somit als Substrat - von DAT, NET und SERT identifiziert werden. Die ausgeprägte Affinität zu DAT und NET suggeriert Missbrauchs- und Abhängigkeitspotential dieser Substanzen. Die eingebetteten Manuskripte offenbaren, dass die Wirkung von Mephedron durch die Phase 1 Metaboliten beeinflusst werden könnte und beinhalten eine pharmakologische Charakterisierung von Substanzen, welche gegenwärtig konsumiert werden (3-FPM) oder zukünftig auf den Drogenmärkten auftauchen könnten (2- und 4-FPM). Weiterst können die angewandten Techniken als Richtlinie betrachtet werden, um die Wirkung von NPS an Monoamintransportern zu entschlüsseln.

 3&&& 

  Declaration####################################################################################################################################### Abstract############################################################################################################################################## Zusammenfassung######################################################################################################################  List of figures#################################################################################################################################%$ Acknowledgements#####################################################################################################################%% Publications arising from this thesis###################################################################################%& Abbreviations################################################################################################################################%) Introduction####################################################################################################################################%, ###############################################################################################################################%, Neurotransmitter transporters#########################################################################################################&$ Monoamine transporters####################################################################################################################&& Physiological role of plasmalemmal monoamine transporters#########################################&( Mode of transport of monoamine transporters########################################################################&- The action of psychostimulants at monoamine transporters#############################################'& Non-transported inhibitors versus amphetamine-like releasing agents###################') New Psychoactive Substances#######################################################################################################(& Aims of this thesis#######################################################################################################################() Materials and Methods###############################################################################################################(* Declaration###############################################################################################################################################(* Publication###############################################################################################################################################(* Results#############################################################################################################################################+- Prologue####################################################################################################################################################+- Publication###############################################################################################################################################,$ Interlude####################################################################################################################################################-' Publication###############################################################################################################################################-( Discussion###################################################################################################################################%&% General discussion#############################################################################################################################%&% Conclusion and future prospects#################################################################################################%&* References###################################################################################################################################%&, Curriculum Vitae########################################################################################################################%() Appendix######################################################################################################################################%(, 

T List of figures

Figure 1: Plasmalemmal and vesicular neurotransmitter transporters Taken from (Gether et al., 2006) (Figure 1). Permission granted by Trends in Pharmacological Sciences (Elsevier).

Figure 2: Illustration of the alternating access model Taken from (Kristensen et al., 2011) (Figure 8A). Permission granted by the American Society for Pharmacology and Experimental Therapeutics (ASPET).

Figure 3: Kinetic scheme of MAT-mediated substrate translocation Created with GraphPad Prism 5.0. The intellectual and graphical input for this figure was derived from (Sitte and Freissmuth, 2015).

Figure 4: Chemical diversity of This figure has been taken from (Sitte and Freissmuth, 2015) (Figure 1). Permission granted by Trends in Pharmacological Sciences (Elsevier).

LK Acknowledgements

At this point I would like to embrace the opportunity to thank my supervisor, Prof. Dr. Harald H. Sitte, who provided continuous support and the latitude to follow my own scientific interests, hence, allowed me to investigate a variety of scientific issues. Further, I am thankful that I was allowed to visit international conferences and that he introduced me to many outstanding scientists.

Further, I would like to thank Prof. Dr. Stefan Boehm, Prof. Dr. Michael Freissmuth and Dr. Michael H. Baumann for their constructive criticism and scientific advice, Marion Holy for her ongoing technical assistance and all my colleagues for the fruitful and humorous discussions and the mutual assistance.

Finally, I would like to express my deepest gratitude to my parents and my family for their longstanding support.

LL Publications arising from this thesis

Research Articles:

Felix P. Mayer, Laurin Wimmer, Ora Dillon-Carter, John S. Partilla, Nadine V. Burchardt, Marko D. Mihovilovic, Michael H. Baumann*, Harald. H. Sitte* (2016); “Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters”; British Journal of Pharmacology, 173(17):2657-68. doi: 10.1111/bph.13547.

Yang Li*, Felix P. Mayer*, Peter Hasenhuetl, Verena Burtscher, Klaus Schicker, Harald H. Sitte, Michael Freissmuth, Walter Sandtner (2017); “Occupancy of the Zinc Binding-site by Transition Metals decreases the Substrate Affinity of the Human by an Allosteric Mechanism“; Journal of Biological Chemistry, doi: 10.1074/jbc.M116.760140

Felix P. Mayer, Nadine V. Burchardt, Ann M. Decker, John S. Partilla, Gavin McLaughlin, Pierce V. Kavanagh, Michael H. Baumann, Bruce E. Blough, Simon D. Brandt, Harald H. Sitte; “Pharmacological characterization of fluorinated phenmetrazine “research chemicals””; In revision, Neuropharmacology (first submission Sept 2016)

Felix P. Mayer, Diethart Schmid, W. Anthony Owens, Georgianna G. Gould, Mia Apuschkin, Oliver Kudlacek, Isabella Salzer, Stefan Boehm, Peter Chiba, Piper H. Williams, Hsiao-Huei Wu, Ulrik Gether, Wouter Koek, Lynette C. Daws*, Harald H. Sitte*; “Organic cation transporter 3: a key player mediating the actions of amphetamine”; Under review, Proceedings of the National Academy of Sciences of the of America (Dec 2016)

Simon Bulling*, Felix P. Mayer*, Diethart Schmid, Walter Sandtner, Harald H. Sitte; “The molecular mechanism of action of mephedrone “; in preparation

LM Felix P. Mayer*, Laurin Wimmer*, Dora Pittrich, Nadine V. Burchardt, Marion Holy, Kathrin Jaentsch, Marko D. Mihovilovic, Harald H. Sitte; “Deciphering the activity of phase I metabolites of mephedrone at monoamine transporters in their enantiopure form“; in preparation

Felix P. Mayer, W. Anthony Owens, Lynette C. Daws, Harald H. Sitte; “The organic cation transporter 3 sheds light on the combination of alcohol and cocaine“; in preparation

Felix P. Mayer, W. Anthony Owens, Lynette C. Daws, Harald H. Sitte; “The synergistic effects of mephedrone and 3,4-methylenedioxypyrovalerone are contingent upon the availability oft he organic cation transporter 3“; in preparation

* denotes equal contribution

Book chapters

Felix P. Mayer, Anton Luf, Constanze Nagy, Marion Holy, Rainer Schmid, Michael Freissmuth, and Harald H. Sitte (2016); “Application of a combined approach to identify new psychoactive street drugs and decipher their mechanisms at monoamine transporters“; accepted for publication, “Neuropharmacology of New Psychoactive Substances (NPS): The Science Behind the Headlines”, Current Topics in Behavioral Neurosciences, doi:10.1007/7854_2016_63

Thomas Steinkellner, Felix P. Mayer, Tina Hofmaier, Marion Holy, Therese Montgomery, Birgit Eisenrauch, Michael Freissmuth, and Harald H. Sitte (2016); “Tracer Flux Measurements to Study Outward Transport by Monoamine Neurotransmitter Transporters“, Neurotransmitter Transporters Investigative Methods, Neuromethods, doi: 10.1007/978-1-4939-3765-3_2

LN Reviews:

Oliver Kudlacek, Tina Hofmaier, Anton Luf, Felix P. Mayer, Thomas Stockner, Constanze Nagy, Marion Holy, Michael Freissmuth, Rainer Schmid, Harald H. Sitte (2017); “Cocaine Adulteration”; accepted, Journal of Chemical Neuroanatomy (Provisionally accepted, subject to minor revisions)

LO Abbreviations  2-FPM 2- fluorophenmetrazine 3-FPM 3- fluorophenmetrazine 4-FPM 4- fluorophenmetrazine 4-MEC 4-methyl-N-ethylcathinone 4-MePPP 4-methyl-α-pyrrolidinopropiophenone 4-OH-mephedrone 4-hydroxytolyl-mephedrone 5-HT Serotonin ADHD deficit hyperactivity disorder AMPH S(+)amphetamine ASD Autism spectrum disorders ATP Adenosine triphosphate AZ Active zone BBB Blood brain barrier C-terminus Carboxyl terminus CaMKII Ca2+/-dependent kinase II CB receptor CNS COMT Catechol-O-methyl-transferase CYP Cytochrome P450 DA Dopamine DAT Dopamine transporter dihydro-mephedrone Dihydro-mephedrone DOPA Dihydroxyphenylalanine EAAT Excitatory amino acid transporter FPM Fluorophenmetrazine FST Forced swim test GABA gamma-aminobutyric acid GAT GABA transporter GLUT Glyt transporter GPCR G- coupled receptor HEK293 cells Human embryonic cells KO Knock out LP LeuT Leucine transporter MAO Monoamine oxidas MAT MDA 3,4-methylendioxyamphetamine MDMA 3,4-methylenedioxymethamphetamine MDPV 3,4-methylenedioxypyrovalerone mephedrone 4-methylmethcathinone, mephedrone MPTP 1-methyl-4phenyl-1,2,5,6-tetrahydropyridine N-terminus Amino-terminus NAc Nucleus accumbens NE Norepinephrine NET Norepinephrine transporter nor-mephedrone 4-methylcathinone NPS New psychoactive substance NRI Norepinephrine reuptake inhibitors NSS Neurotransmitter sodium:symporter NTT Neurotransmitter transporter OCT Organic cation transporter PD Parkinson’s Disease

PIP2 Phosphatidylinositol-4,5-bisphosphate PK Protein kinase PKC Protein kinase C PKG Protein kinase G PMAT Plasmalemmal monoamine transporter SERT SLC Solute carrier SNRI Serotonin norepinephrine reuptake inhibitors SSRI Selective serotonin reuptake inhibitors TCA Tricyclic TH hydroxylase THC Δ9-tetrahydrocannabinol TMD Transmembrane domain TST Tail suspension test VAChT Vesicular transporter

LQ VMAT Vesicular monoamine transporter VTA

LR Introduction

Neurotransmission

Manifold and complex intercellular communication constitutes a hallmark of the nervous system. To maintain the nervous system at a functional state, the communication between individual requires delicate regulation and demands both flexibility and precision (Bermingham and Blakely, 2016). Intercellular exchange of signals occurs at distinct anatomical connections via electrical or chemical synapses (Pereda, 2014). Electrical signaling requires direct connections between cells (Pereda, 2014). Hence, electrical synapses allow for rapid communication via ionic channels, i.e. gap junctions (Szczupak, 2016), and a synaptic delay is virtually absent (Kandel and Schwartz, 2000). However, despite evidence supporting the plasticity of electrical synapses (O'Brien, 2014), the mechanism for modulating the underlying nature of the electrical signal remains elusive, i.e. hyperpolarizing/depolarizing synaptic membranes (Kandel and Schwartz, 2000, Freissmuth et al., 2012). In contrast, chemical synapses convert the arriving electrical signals into chemical signals. Hence, in chemical synapses specific molecules, i.e. , facilitate the communication between pre- and post-synaptic termini (Valenstein, 2002). Originally discovered by Otto Loewi at the beginning of the 20th century (Loewi, 1921, Zimmer, 2006), signaling molecules are released into the extracellular compartment and exert their action at their cognate receptor targets. This revolutionary concept that chemicals are capable of transmitting a signal was highly controversial. However, in time the accumulated scientific evidence in favor of chemical neurotransmission led to the awarding of the Nobel Prize to Otto Loewi and Henry Dale in 1936 (Valenstein, 2002). The regulated release of neurotransmitters depends on the fusion of synaptic vesicles to the plasma membrane in the active zone (AZ) of the presynaptic bouton (Gundelfinger et al., 2015) in a calcium dependent manner (Sudhof, 2012). Upon the arrival of an action potential, the presynaptic specialization depolarizes and voltage- gated calcium channels are activated allowing calcium influx into the AZ. The rise in cytosolic calcium from <100 nM up to concentrations in the millimolar range promotes and the release of neurotransmitters (Freissmuth et al., 2012).

LS The synaptic machinery involved in this complex process is composed of SNARE that are located on vesicles (vSNARES) and the plasma membrane (tSNAREs), i.e. synaptobrevin residing on vesicular membranes and plasmalemmal syntaxin-1 and SNAP-25. The interaction of v- and tSNAREs tethers the vesicles to the membrane. Binding of calcium to the calcium-sensor synaptotagmin and complexins initializes the fusion of the vesicular and the plasmalemmal membranes (Kandel and Schwartz, 2000, Aktories et al., 2009). Subsequently, neurotransmitters that have been stored within the synaptic vesicles diffuse into the synaptic cleft and activate receptors located on postsynaptic neurons and autoreceptors, present on the releasing neurons (Kandel and Schwartz, 2000). The receptors may be further subdivided into i) transmitter gated channels and ii) G-protein-coupled receptors (Kandel and Schwartz, 2000, Freissmuth et al., 2012). Upon binding of neurotransmitters, ionotropic receptors, i.e. transmitter-gated ion channels, undergo conformational changes and open. Depending on the ion selectivity of the respective receptor, ionic fluxes can result in de- or hyperpolarization (Aktories et al., 2009). For instance, binding of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to the GABAA receptor results in profound influx of Cl- and concomitant hyperpolarization, which exerts inhibitory effects on the target (Boehm and Kubista, 2002, Freissmuth et al., 2012). The metabotropic G-protein coupled receptors (GPCRs) mediate their effects via second messengers and activation of signal cascades. Thus, as opposed to ionotropic receptors, GPCRs mediate their effects with a temporal delay, but enable manifold and plastic communication between neurons by influencing different signal pathways. Remarkably, the observation that receptors define the cellular response to a given neurotransmitter or drug dates back to the early 20th century: John Newport Langley observed that can cause both contraction and relaxation in different organs (Langley, 1921, Valenstein, 2002).

LT Neurotransmitter transporters

Three main mechanisms have been proposed to terminate the activity of neurotransmitters in the synaptic cleft (Iversen, 1971): These include i) enzymatic conversion of the neurotransmitter into pharmacologically inactive metabolites ii) diffusion and subsequent dilution of the neurotransmitter, and iii) transporter mediated clearance. This latter process is based on specific transporters that sequester neurotransmitters from the extracellular compartment and mediate the active transport of their substrates into glial or neuronal cells (Masson et al., 1999). In neuronal cells, vesicular transporters further concentrate the re-captured neurotransmitters into synaptic vesicles to maintain and replenish the releasable pool of neurotransmitters (Masson et al., 1999). The broad chemical spectrum of neurotransmitters led to the evolution of a variety of specific neurotransmitter transporters (NTTs), ranging from specific carrier proteins for glutamate to high-affinity transporters for glycine, GABA, dopamine (DA), serotonin (5-HT) and norepinephrine (NE) (Nelson, 1998). NTTs can be divided into major subclasses, i.e. solute carrier (SLC) families, with the SLC1 and SLC6-family being the two major subfamilies for plasmalemmal NTTs (Gether et al., 2006). The SLC1 family comprises transporters for excitatory amino acids (EAATs), i.e. glutamate (Focke et al., 2013, Grewer et al., 2014), whereas the SLC6 family harbors a broader spectrum of transporters for chemically diverse substrates, e.g. GABA, glycine, proline, DA, 5-HT and NE (Kristensen et al., 2011) (Figure 1). In addition to the SLC1 and SLC6 family members, which translocate their substrates across the plasma membrane, intracellular transporters accumulate neurotransmitters into synaptic vesicles. These transporters can be classified as vesicular glutamate (SLC17), monoamine (SLC18) and inhibitory amino acid (SLC32) transporters (Gether et al., 2006) (Figure 1). The well-orchestrated transport events by plasmalemmal and vesicular transporters maintain neurotransmitter homeostasis by regulating the activity of neurotransmitters in the extracellular space and by shaping the releasable pools (Blakely and Edwards, 2012). Consequently, NTTs serve as valuable targets for the treatment of neurological and psychiatric disorders that arise from imbalances in the synaptic tone of specific neurotransmitters, including epilepsy and depression. However, NTTs also serve as the primary

MK molecular targets for a variety of recreationally abused substances, such as cocaine or amphetamines (Gether et al., 2006, Broer and Gether, 2012).

Figure 1: Plasmalemmal and vesicular neurotransmitter transporters. a) Glutamate-releasing neurons express transporters of the SLC17 family that are located to vesicles (green, vGLUT1-3) to sustain a releasable pool of glutamate. Exocytically released glutamate interacts with -gated ion channels, i.e. ionotropic receptors, or GPCR-coupled receptors to exert its effects. Plasmalemmal glutamate transporters of the SLC1 family (EAAT1-5, blue) reside on pre- and postsynaptic termini and adjacent glial cells to sequester extracellular glutamate. b) Pre-synaptic neurons harboring DA, 5- HT, NE, GABA or glycine express VMATs (SLC18), vesicular inhibitory amino acid transporters (VIAATs, SLC18) or vesicular acetylcholine transporters (VAChTs, SLC32) to accumulate the respective neurotransmitter into synaptic vesicles (green). Extracellular neurotransmitters interact with GPCR-coupled or ligan-gated ion channels receptors to affect their target cells. SLC6-transporters at the plasma membrane clear the vicinity of neurotransmitters (red). DAT, NET, SERT, glycine transporter 2 (GlyT2), and the GABA transporters 1 and 2 (GAT1 and 2, respectively) reside on the presynaptic neuron. Glial cells express GlyT1, and GAT1, GAT2 and GAT3. This figure has been taken from (Gether et al., 2006).

ML Monoamine transporters

As outlined in the previous section, the transporters for the monoamines DA, 5-HT and NE fall into the SLC6 family (Kristensen et al., 2011). Like other members of the SLC1 and SLC6 family, the transporters for DA (dopamine transporter, DAT; SLC6A3), 5-HT (serotonin transporter, SERT; SLC6A4) and NE (norepinephrine transporter, NET; SLC6A2) utilize the sodium gradient as driving force for their concentrative uptake process (Rudnick and Clark, 1993). This coupling of energetically unfavorable “uphill” transport of neurotransmitters to energetically favorable “downhill” transport of (Rudnick, 1998) allows for neurotransmitter translocation. Consequently, these transporters are also referred to as neurotransmitter:sodium symporters (NSS) (Saier et al., 2006, Sitte and Freissmuth, 2010) or Na+/Cl- dependent transporters (Nelson, 1998, Broer and Gether, 2012) and constitute secondary active transporters (Chen et al., 2004, Kristensen et al., 2011). They strictly depend on the activity of Na+,K+-ATPase, which generates the gradients of potassium and sodium (Sitte and Freissmuth, 2010, Sitte and Freissmuth, 2015) by transporting three Na+ ions out of the cell and two K+ ions into the cells per hydrolyzed adenosine triphophosphate (ATP) molecule (Rice et al., 2001). On a cellular level, DAT, NET and SERT are located at perisynaptic sites (Torres et al., 2003). Anatomically positioning these monoamine transporters (MATs) to the pre- synaptic nerve terminal allows not only for the fastest, but also for the most economical means to terminate synaptic transmission (Sitte and Freissmuth, 2010) . Experimental evidence revealed that the MATs at the plasma membrane are physically linked to vesicular monoamine transporters (VMATs) via adapter proteins (Egana et al., 2009). The close proximity ensures the recycling of previously released neurotransmitters into vesicular storage pools. As the turnover number of VMAT2 (Peter et al., 1994) exceeds the turnover number of the plasmalemmal MATs (Sonders et al., 1997, Erreger et al., 2008, Sucic et al., 2010, Kristensen et al., 2011), monoamines are rapidly packed into vesicles and their duration within the cytosol is kept at a minimum (Sitte and Freissmuth, 2015). Monoamines are promptly degraded by means of monoamine oxidases A and B (MAO-A and B, respectively) and catechol-O-methyl-transferases (COMTs) (Godar and Bortolato, 2014). It is worth pointing out that enzymatic degradation of cytosolic monoamines results in potentially neurotoxic byproducts (Bortolato et al., 2008), which inevitably highlight the

MM importance of VMATs for maintaining monoaminergic neurons at a viable state (Lin et al., 2010, Lin et al., 2011). Structurally, as members of the SLC6 family, MATs share a membrane topology consisting of 12 transmembrane domains (TMDs), a large extracellular loop between TMD 3 and 4 (See Figure 1) containing glycosylation consensus sites, intracellular carboxyl- and amino-termini (C- and N-terminus, respectively) and typically consist of 617 to 632 amino acids (Gether et al., 2006, Kristensen et al., 2011). The activity of NTTs is subject to complex and interwoven regulatory mechanisms, which include trafficking to and from the plasma membrane, and glycosylation (Mortensen and Amara, 2003, Kristensen et al., 2011, Blakely and Edwards, 2012, Bermingham and Blakely, 2016). Remarkably, the C-and N-terminal domains of the NTTs from the SLC6 family are highly diverse and have been proposed to provide the basis for transporter-specific regulation by proteins interacting with these domains (Kristensen et al., 2011, Blakely and Edwards, 2012, Bermingham and Blakely, 2016). A growing body of experimental evidence supports this hypothesis. For example, activation of protein kinase (PK) C (PKC) increases DAT phosphorylation at its N-terminus and decreases DAT activity (Vaughan et al., 1997, Foster et al., 2002). This reduction in dopamine uptake has been shown to result from decreased levels of DAT at the cell surface (Daniels and Amara, 1999). In addition, Ca2+/calmodulin-dependent kinase II (CaMKII) mediated phosphorylation of the DAT N-terminus impinges on DAT function in psychostimulant action (Fog et al., 2006, Steinkellner et al., 2012, Steinkellner et al., 2015). Further, the internalization rates of NET and DAT are regulated by specific elements in their N-termini (Vuorenpaa et al., 2016). The expression pattern of MATs is highly correlated with the corresponding monoaminergic systems in nervous system. This can be attributed to the finding that MATs are virtually non-existent in neurons that do not synthesize the cognate neurotransmitter (Torres et al., 2003). Consequently, high expression levels of NET are observed within the , DAT levels are high in the ventral tegmental area (VTA) and the substantia nigra (Hoffman et al., 1998, Torres et al., 2003) and SERT is readily abundant in dorsal and median raphe nuclei and the hippocampus (Sur et al., 1996).

MN Physiological role of plasmalemmal monoamine transporters

The monoamines DA, NE and 5-HT are involved in the regulation of a variety of physiological functions and emotional states, including appetite, motivation, reward, aggression, motor control, and sleep (Chen et al., 2004). The essential role of the corresponding MATs in shaping and regulating the strength or the duration of monoaminergic signalling and the underlying phenotypic manifestations has been conclusively substantiated in studies using pharmacological tools and genetic ablation of the individual transporters (Kristensen et al., 2011). Furthermore, polymorphisms in the encoding for DAT, NET or SERT have been associated with clinically adverse , such as attention deficit hyperactivity disorder (ADHD), depression, anxiety and autism spectrum disorders (ASD) (Masson et al., 1999, Kristensen et al., 2011, Blakely and Edwards, 2012, Hansen et al., 2014, Wu et al., 2015). Moreover, removal of a transporter for large neutral amino acids, i.e. SLC7A5, from the blood brain barrier (BBB) also results in ASD-related symptoms (Tarlungeanu et al., 2016), which highlights the critical relevance of different families of transport proteins. Remarkably, the vast importance of transporter-mediated neurotransmitter clearance became evident more than 50 years ago. In the early 1960s it was found that sympathetic tissue accumulates tritiated-NE ([3H]-NE) (Whitby et al., 1961) and that this uptake could be inhibited by the tricyclic antidepressant (TCA) (Axelrod et al., 1961). These observations provided the first insights into the molecular mechanism underlying the effects of clinically used TCAs. Later, it was discovered that the TCAs imipramine and amitryptiline were potent inhibitors of both 5-HT and NE-uptake, which further strengthened the hypothesis that depression is linked to a deficiency in monoamines (Iversen, 2000). In mice lacking DAT, the most striking is spontaneous hyperlocomotion (Giros et al., 1996) and a reduction in bodyweight (Kalueff et al., 2007). Neurochemical characterizations of DAT-knock out (KO) mice revealed that extracellular DA was elevated in the . As determined by real time measurements, due to the drastically reduced DA-clearance rates, DA persisted up to 300 times longer in the extracellular compartment than in wild-type controls (Giros et al., 1996, Jones et al., 1998a, Kristensen et al., 2011). Remarkably, genetic deletion of DAT is accompanied by adaptive changes. In absence of DAT,

MO intratissular DA levels were found to be reduced 20-fold and the amount of intraneuronal DA mainly stems from a newly synthesized pool rather than vesicular stores (Gainetdinov, 2008). The latter statement further fuels the hypothesis that the plasmalemmal and vesicular MATs operate in an economical relay to re-capture and recycle monoamines (Gainetdinov and Caron, 2003, Gainetdinov, 2008, Sitte and Freissmuth, 2015). To compensate for the drastically elevated tone, DA receptors were generally found to be down regulated and desensitized (Giros et al., 1996, Jones et al., 1999, Gainetdinov and Caron, 2003, Gainetdinov, 2008). In addition to the altered locomotor activity, DAT-KO mice show abnormal, i.e. reduced, habituation upon exposure to novel environments and exhibit deficits in behavioral models (Gainetdinov, 2008, Kristensen et al., 2011). Mice devoid of NET (NET-KO) demonstrated higher levels of extracellular NE and reduced NE-clearance rates (Xu et al., 2000). NET-KO mice mirrored the finding from DAT-KO mice, i.e. reduced intraneuronal storage pools of NE and electrical stimulation yielded blunted NE release (Gainetdinov and Caron, 2003). In behavioral assays, absence of NET was linked to an “antidepressant-treated” phenotype. In the tail-suspension and forced swim test (TST and FST, respectively), mice are confronted with an inescapable situation. Since a mouse attempts to escape this situation, the time spent immobile is thought to directly correlate with depression-like behavior (Petit-Demouliere et al., 2005, Can et al., 2012). In both, the TST and FST, NET-KO mice exhibited escape-oriented behaviors to the same extent as their wild type counterparts that have been treated with (Xu et al., 2000). Furthermore, absence of NET is associated with , elevated blood pressure upon sympathetic activation (Kristensen et al., 2011) and a reduction in bodyweight (Kalueff et al., 2007). As observed in DAT- and NET-KO mice, disruption of the gene coding for SERT (SERT-KO) in mice (Bengel et al., 1998) results in elevated 5-HT levels in the extracellular space and reduced intratissular 5-HT (Bengel et al., 1998, Chen et al., 2004, Gardier, 2009). With respect to receptors, an adaptive down regulation of the

5-HT1A receptor has been described in the dorsal raphe nucleus (Gardier, 2009). In SERT-KO mice, the perturbation of 5-HT homeostasis manifested in enhanced stress- and anxiety-related behaviors. For instance, SERT-KO mice show less explorative behavior (Kristensen et al., 2011) in the elevated plus maze test. In this test, mice are placed onto an apparatus that consists of two open and two enclosed

MP arms. The time spent, i.e. duration and entries, in the open arms is considered as a measure for non-anxious and explorative behavior (Walf and Frye, 2007).

The vesicular monoamine transporters VMAT1 and VMAT2 account for the packaging of monoamines into synaptic vesicles (Guillot and Miller, 2009, Lohr et al., 2016). Both VMAT-isoforms are members of the SLC18-family (VMAT1, SLC18A1; VMAT2, SLC18A2) and harness the acidic milieu in synaptic vesicles as driving force to accumulate their substrates by acting as monoamine/proton antiporters (Rudnick et al., 1990, Gether et al., 2006, Lawal and Krantz, 2013). While VMAT1 is mainly found in neuroendocrine cells, VMAT2 is readily expressed in monoaminergic neurons of the central nervous system (CNS) (Hoffman et al., 1998, Lohr et al., 2016) as well as the peripheral and enteric nervous system (German et al., 2015). The rat VMAT2 protein consists of 515 amino acids (Erickson et al., 1992) and contains 12 TMDs with cytosolic N- and C-Termini. Analogous to plasmalemmal MATs, the terminal regions of VMAT2 are implicated in regulating VMAT2 function and contain phosphorylation sites (German et al., 2015). The indispensable role of VMAT2 in neurotransmission is emphasized by the fact that VMAT2-KO mice die within the first two weeks after birth (Wang et al., 1997, Fon et al., 1997). In contrast to mice fully lacking VMAT2, VMAT2 heterozygous mice (VMAT2+/-) are viable and reach adulthood (Wang et al., 1997, Fon et al., 1997, Lin et al., 2011). VMAT2+/- mice show a 50% reduction in VMAT2 protein levels in striatum and substantia nigra and markedly reduced release of DA upon depolarization. A gene-dose effect is visible in the reduction in whole brain monoamines. However, this effect did not reach statistical significance in VMAT2+/- mice (Wang et al., 1997). VMAT2-KO mice are essentially devoid of monoamines in brain and DA-release upon electrical stimulation is virtually absent (Wang et al., 1997, Fon et al., 1997). Surprisingly, recombination events led to the generation of mice with a 95 % decrease in VMAT2 expression (VMAT2-LO) (Mooslehner et al., 2001). The relative lack of VMAT2 in these mice drastically reduces the monoamine content in synaptic vesicles by 80 % (Lohr et al., 2016) and the levels of DA, NE and 5-HT are reduced throughout the brain (Mooslehner et al., 2001, Caudle et al., 2007, Lin et al., 2011, Lohr et al., 2016). VMAT2+/- and VMAT2-LO mice are both hypersensitive to psychostimulants and toxicants, such as 1-methyl-4phenyl-1,2,5,6-tetrahydropyridine

MQ (MPTP) (Javitch et al., 1985, Wang et al., 1997, Mooslehner et al., 2001, Lohr et al., 2016), which is used to destroy dopaminergic neurons to induce symptoms of Parkinson’s Disease (PD) in the MPTP-model (Porras et al., 2012). Reductions in VMAT2 are associated with neurodegeneration, PD (Takahashi et al., 1997, Caudle et al., 2007, Eiden and Weihe, 2011, Taylor et al., 2014) and correlate with age- related changes in the dopaminergic system (Hall et al., 2014). In contrast, elevated VMAT2 expression exerts protective effects and promotes cell survival (Lohr et al., 2016). These findings are in alignment with the hypothesis that cytosolic DA is cytotoxic and that mechanisms that reduce DA in the cytosol are neuroprotective (Mosharov et al., 2009). This notion is further supported by the observation that tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, two enzymes implicated in the synthesis of the DA, NE and adrenaline (Kandel and Schwartz, 2000, Freissmuth et al., 2012), have been found to interact with VMAT2 containing vesicles (Cartier et al., 2010, Lin et al., 2011). Hence, the limited spatial separation is believed ensure the immediate sequestration of newly synthesized catecholamines (Cartier et al., 2010, German et al., 2015). These insights emphasize the pivotal role of VMAT2 in monoaminergic neurotransmission and in maintaining neurons at a vital state (Lin et al., 2011). Genetic model organisms that are devoid of MATs fuelled our understanding of monoaminergic signalling. However, genetic manipulations are often accompanied by adaptive changes, as such, these observations should be treated and interpreted with care. Nevertheless, targeting MATs by pharmacological means further supports the essential role of carrier mediated monoamine reuptake. MATs are well- established targets of anti- and psychostimulants (Gainetdinov and Caron, 2003). Results obtained from pharmacological studies closely resemble those obtained from studies in MAT-KO mice (Lin et al., 2011). For instance, administration of the selective DAT-inhibitor GBR-12935 stimulated locomotion in mice (Tolliver and Carney, 1994). SERT and NET are targeted by clinically applied anti-depressants (Sitte and Freissmuth, 2010, Kristensen et al., 2011). The first generation of TCAs was characterized by moderate selectivity for SERT over NET or vice versa with remarkable affinities for targets in the CNS other than MATs (Iversen, 2000). On-going research led to the development of more specific inhibitors and reduced off-target effects. Clinically used monoamine re- uptake inhibitors may favor SERT (selective serotonin reuptake inhibitors, SSRIs) or

MR NET (NRIs) or target SERT and NET with similar affinities (SNRIs). All three classes reveal anti- properties. However, the successful application of fluoxetine, a SSRI, swung the focus towards agents targeting the SERT for the treatment of anxiety and depression (Iversen, 2000, Rudnick et al., 2014). NRIs are primarily used to ameliorate the symptoms of ADHD (Kristensen et al., 2011). Additionally, the rewarding and addictive effects many drugs of abuse, including cocaine, have been attributed to their abilities to interact with MATs, with an emphasis on their interaction with the DAT (Gether et al., 2006, Broer and Gether, 2012).

MS Mode of transport of monoamine transporters

It is generally believed that transporters undergo conformational rearrangements to translocate their substrates across biological membranes (Penmatsa and Gouaux, 2014) . According to the alternating access model, which was proposed 50 years ago (Jardetzky, 1966), the transporter is only accessible from one site of the membrane at any given time point (Penmatsa and Gouaux, 2014). Importantly, this property essentially differentiates transporters from channels (Sitte and Freissmuth, 2010). However, due to the observed magnitude of transporter-associated currents, it has been proposed that MATs may also adopt a channel-like mode (Sonders and Amara, 1996, DeFelice and Goswami, 2007). During the transport cycle, the transporter sequentially moves from an “outward- facing” conformation to an “inward-facing” conformation via structural intermediates, i.e. occluding states (Kristensen et al., 2011, Focke et al., 2013) (Figure 2). In the outward-facing open conformation, the substrate-binding pocket is exposed to the extracellular milieu to retrieve extracellular substrates and co-substrates. In the inward-facing open conformation the binding pocket opens up towards the cytosol, allowing the captured substrates to reach the intracellular compartment. The occluded states represent conformations in which both gates to the binding pocket are closed (Pramod et al., 2013). The occluded states per se were not proposed in the original alternating access model by Jardetzky et al. (Jardetzky, 1966, LeVine et al., 2016). However, Jardetzky et al. already predicted a transition state between the outward- and inward-open conformations with both gates being inaccessible (Sitte and Freissmuth, 2010).

MT

Figure 2: Illustration of the alternating access model. To shuttle substrates across the membrane, the transporter adopts various conformations. In the outward-facing open conformation, extracellular substrate and co-substrate enter the . Via occluded states, the transporter adopts the inward-facing open conformation and releases the cargo into the cytosol and engages counter transported ions, e.g. potassium for SERT (Schicker et al., 2012) or substrates. This figure has been taken from (Kristensen et al., 2011).

In a simplified kinetic scheme, following the binding of the co-substrates sodium and chloride ions, the substrate occupies the substrate-binding pocket of the MAT in the outward-facing conformation. As the outer gate closes, the transporter adopts the substrate-bound occluded state and transitions to the inward-facing conformation to release the substrate and the ions into the cytosol. Subsequently, to fulfill the cycle, the transporter moves back to the outward-facing conformation via an occluded empty state (Pramod et al., 2013, Sitte and Freissmuth, 2015) (Figure 3). In the case of SERT, the movement from the inward-facing conformation to the outward-facing conformation via the occluded empty state requires the presence of potassium (Schicker et al., 2012, Sitte and Freissmuth, 2015, Hasenhuetl et al., 2016), which differentiates it from its close relatives DAT and NET (Rudnick, 1998).

NK

Figure 3: Kinetic scheme of MAT-mediated substrate translocation. Scheme representing the interaction of substrate (S) with the transporter (T) in the outward-facing (o) or inward-facing (i) conformation. The occluding states (occ) represent conformations in which access to the binding site is prevented. The transporter adopts the conducting state (Cond) via inward-facing conformation. For detailed description of the transport cycle see (Schicker et al., 2012). The intellectual and graphical input for this figure was derived from (Sitte and Freissmuth, 2015).

The advent of X-ray crystal structures of a bacterial NSS-homologue led to a giant leap in the understanding of the transport cycle of NSS. Remarkably, the snapshots obtained from various conformations strengthened the concept of the alternating access model (Sitte and Freissmuth, 2010). In 2005, the X-ray crystal structure of the leucine transporter (LeuT) from Aquifex aeolicus was reported (Yamashita et al., 2005). LeuT and NSS share approximately 20-25% overall sequence identity (Kristensen et al., 2011), with more than 50% of the residues being identical around the binding site (Pramod et al., 2013). Akin to mammalian MATs, LeuT comprises 12 TMDs and intracellular N- and C-termini (Yamashita et al., 2005), albeit the termini of the bacterial LeuT are much shorter than the termini of mammalian MATs (Pramod et al., 2013). The tertiary structure of LeuT provided excellent insights into the architecture of the binding sites for substrates and ions of NSS, and, importantly, the molecular rearrangements of the protein during the transport cycle (Kristensen et al., 2011). Recently, crystal structures of the DAT from Drosophila melanogaster bound to a TCA (Penmatsa et al., 2013) and psychostimulants (Wang et al., 2015) and human SERT bound to paroxetine and (S)-citalopram (Coleman et al., 2016) have

NL been reported. These novel reports unravel the molecular interactions of psychoactive agents with MATs and provide templates for the development of compounds with improved selectivity profiles.

The action of psychostimulants at monoamine transporters

Various agents, including clinically relevant and abused compounds, exert their effects by targeting MATs (Kalueff et al., 2007). Through evidence provided by the use of pharmacological and genetic tools, disruption of MAT-function brings forth an increase in extracellular monoamine concentration and results in profound and continuous stimulation of target neurons (Torres et al., 2003). Epitomized by the clinical application of antidepressants and anxiolytics, rational and prudent manipulations of MAT-function provide precious means to ameliorate symptoms that arise from imbalances in monoaminergic signaling. However, MATs also serve as a target for a plethora of recreationally used substances, i.e. psychostimulants, such as cocaine or (“crystal meth”) (Kristensen et al., 2011). These drugs pose potential health risks (Baumann and Volkow, 2016) and are associated with a high risk of addiction (Kalivas, 2007). Consequently, psychostimulant-use disorders constitute a major burden for the public health on a global scale and, unfortunately, the treatments for psychostimulant addiction available at present are far from being satisfying (Phillips et al., 2014).

Interestingly, psychostimulants, such as amphetamine, methamphetamine or MDMA might be considered as synthetic and rather “newer” drugs. However, psychostimulants have already been consumed for thousands of years (Sulzer, 2011). For instance, cocaine is naturally found in Erythoxylon coca, a plant that has been cultivated for its psychoactive ingredient (Sulzer et al., 2005, Jay, 2015). Motivated by Sigmund Freud’s enthusiastic reports about cocaine (Sulzer et al., 2005, Freud, 1884), Carl Koller introduced cocaine as a local anesthetic into modern medicine (Honegger and Hessler, 1970, Markel, 2011), since cocaine does not only target MATs but also inhibits sodium channels at higher concentrations (Kyle and Ilyin, 2007).

NM The versatile class of amphetamine-like substances also contains naturally occurring compounds that have been appreciated for their energizing effects. Herbal products of Ephedra sinica and Catha edulis contain and , respectively. The herbs can be chewed or are consumed as tea (“Mormon Tea”) and early reports on the use of Ephedra sinica products date back to the first century AD (Sulzer et al., 2005, Glennon, 2014). Nevertheless, S(+)amphetamine (AMPH, “speed”), 3,4- methylendioxyamphetamine (MDA) and MDMA, are completely synthetic drugs that do not occur naturally. AMPH, MDMA and MDA were synthesized around 1900 and patented as tranquilizers and appetite or cough suppressants (Kalant, 2001). Owing to the properties, amphetamines were administered to soldiers during the second World War (Lemere, 1966, Kalivas, 2007) and gained popularity amongst students, artists, truck drivers and musicians (Sulzer et al., 2005).

On a molecular level, psychostimulants that interact with MATs prolong the dwelling time of monoamines in the extracellular space. As such, they can also be classified as indirectly acting. Early investigations showed that some compounds raise blood pressure - akin to innervation by the sympathetic system. Therefore, these compounds were categorized as sympathomimetics (Barger and Dale, 1910). Later studies revealed that some compounds, i.e. directly acting sympathomimetics, were insensitive towards reserpine and still exerted membrane contraction after sympathetic denervation. In stark contrast, various indirectly acting sympathomimetics were sensitive towards reserpine and failed to cause contraction upon denervation (Fleckenstein and Burn, 1953, Burn and Rand, 1958). On the basis of these findings, Burn and colleagues hypothesized that the reserpine-sensitive compounds release a “noradrenaline-like” substance (Burn and Rand, 1958, Sulzer, 2011), whereas directly acting sympathomimetics act as at adrenoreceptors without elevating the extracellular NE concentration (Docherty, 2008). Further inquiry into the mechanism of action of indirectly acting sympathomimetics arose from the observations that [3H]NE was concentrated into synaptic nerve terminals (Whitby et al., 1961) and that the psychostimulants cocaine (Whitby et al., 1960) and amphetamine (Axelrod et al., 1961) markedly reduced the uptake of [3H]NE into these terminals. In the following years, SERT and DAT were identified as additional and fundamental targets of cocaine and amphetamines in addition to NET (Amara and Kuhar, 1993, Rudnick, 1998).

NN The original observations date back more than 50 years. However, unraveling the molecular underpinnings of drug-induced elevations of extracellular monoamines turned out to be a surprisingly challenging task. For the sake of simplicity, the terms “psychostimulant” and “stimulant” will now be used to refer to drugs that interact with MATs, rather than monoamine receptors. Drugs that target MATs can act in two different ways: i) as “classical” re-uptake inhibitors (“blockers”) or as ii) substrate-type “releasers”. Briefly, the aforementioned “blockers” simply inhibit the transporter-mediated re-uptake of extracellular monoamines. In contrast, “releasers” cause a transporter-mediated “efflux” of monoamines. Despite the two classes of psychostimulants differ in the molecular mode of action; both types elevate the synaptic concentration of monoamines (Sitte and Freissmuth, 2015). An increase in extracellular monoamines is associated with reward (Sulzer, 2011, Robison and Nestler, 2011, Russo and Nestler, 2013, Luscher, 2016). Hence, drugs that reinforce the reward-circuitry (Russo and Nestler, 2013) may also be considered as chemical replacements for natural rewarding stimuli, such as food intake or sex (Di Chiara, 1995). This feature appears to be attributable to profound effects on dopaminergic neurotransmission (Cami and Farre, 2003, Sulzer, 2011, Taylor et al., 2013). It is generally perceived that addictive drugs activate the reward circuitry, which comprises midbrain dopaminergic neurons in the VTA, including their projections to the nucleus accumbens (NAc), dorsal striatum, amygdala and various regions of the (Robison and Nestler, 2011, Russo and Nestler, 2013). It is not surprising that psychostimulants that increase the availability of DA in the extracellular space can cause addiction and severe drug seeking behaviors (Volkow, 2005, Kalivas, 2007, Sulzer, 2011). The hypothesis that DA is essentially involved in addiction is further strengthened by the finding that the addictive properties of a drug correlate with its to inhibit [3H] binding to DAT (Ritz et al., 1987, Amara and Sonders, 1998). Interestingly, it has been pointed out that many psychostimulants lose the ability to affect DA clearance in mice lacking DAT (Gainetdinov and Caron, 2003). However, at that point, it is important to mention that DAT-KO mice self-administer cocaine (Rocha et al., 1998) and develop place preference for cocaine, whereas the latter phenomenon is lost in mice lacking DAT and SERT (Sora et al., 2001). Furthermore, amphetamine and cocaine elevate DA in the NAc of DAT-KO and wild-type mice (Carboni et al., 2001). Additionally, an

NO essential role as mediator of the hedonic effects of addictive drugs has been attributed to NE (Jasmin et al., 2006). Together, despite compelling evidence highlights the implication of DA in addiction; these findings support the notion that addictive and rewarding effects of psychostimulants are not solely mediated by their interaction with DAT.

Further effects range from intensified , bolstered self-esteem and to the suppression of appetite and the need for sleep, and may even include empathy (Romanelli and Smith, 2006, Sitte and Freissmuth, 2015, Liechti, 2015). In the periphery, psychostimulants may cause , increased muscle-tension, tachypnea or (Phillips et al., 2014). Prolonged abuse can result in and restlessness. Higher doses can trigger , stereotyped behaviors and eventually even cause respiratory failure, cardiac , or infarct (Gay, 1982, Phillips et al., 2014).

Non-transported inhibitors versus amphetamine-like releasing agents

As mentioned briefly in the previous section, psychostimulants can be classified according to their action at MATs, i.e. as “releasers” and “blockers” (Sandtner et al., 2016). The latter act as non-transported re-uptake inhibitors, like cocaine, and elevate the extracellular concentration of monoamines by blocking the transporter- mediated re-uptake (Sitte and Freissmuth, 2010). In MATs, the binding sites for re- uptake inhibitors and the physiological substrates may overlap (Beuming et al., 2008, Bisgaard et al., 2011, Penmatsa and Gouaux, 2014), hence, most uptake inhibitors act as competitive inhibitors (Sandtner et al., 2016). Uptake inhibitors perturb the natural function of MATs, i.e. the re-uptake of the respective monoamine, by stabilizing the transporter in a specific conformation and thereby arresting the transport cycle (Beuming et al., 2008, Singh et al., 2008, Zhou et al., 2009, Penmatsa and Gouaux, 2014, Wang et al., 2015). As cocaine-like inhibitors merely prevent the re-uptake of extracellular substrates, it is evident that exocytotic release of monoamines is a prerequisite for the action of inhibitors.

NP In contrast, amphetamine-like “releasers” are capable of triggering “transporter- mediated” release of monoamines, independently from exocytosis. Carrier-mediated release is also referred to as non-exocytotic release (Sitte and Freissmuth, 2015). Releasers, such as S(+)amphetamine (also referred to as D-amphetamine), are accepted as substrates by MATs and actively transported into the cytosol (Bonisch, 1984, Sitte et al., 1998). Consequently, releasers extrude the physiological substrates from the binding sites and act as competitive inhibitors (Sitte and Freissmuth, 2015). However, the identity-establishing feature of releasers is their ability to invert the natural transport direction of MATs (Sandtner et al., 2016). As a net-result, “inverted” transporters capture cytosolic monoamines and release them into the extracellular space. At this point it is worth mentioning that every MAT-substrate bears the potential to induce transporter-mediated release (Pifl et al., 1995, Sitte et al., 1998, Scholze et al., 2000). In keeping with the “alternating access model”, any substrate that is transported into the cytosol gives rise to transporters in the inward-facing conformation, which are then accessible for cytosolic substrates. Consequently, substrates may engage transporters in the inward-facing conformation and are subsequently released into the extracellular space as the transporter moves back to the outward-facing conformation. However, this “facilitated exchange diffusion model” (Fischer and Cho, 1979, Trendelenburg et al., 1987) is difficult to reconcile with the physiological role of the transporters. In fact, if this phenomenon holds true in vivo, MATs would fail to terminate monoaminergic signaling and the presence of their substrates would simply result in the futile cycling of these transporters (Sitte and Freissmuth, 2010). As an alternative to the facilitated exchange diffusion model, it has been proposed that efflux might occur via “channel like transporters”. DAT (Sonders et al., 1997, Sitte et al., 1998), NET (Galli et al., 1996) and SERT (Galli et al., 1997) can exhibit channel-like electrical properties. Solely driven by the membrane potential and the electrochemical gradient, a channel-like mode would allow a random number of substrates to pass across the membrane via the transporter (DeFelice and Goswami, 2007). Consequently, a channel-mode has been suggested to envisage spike-like increases of DA in the presence of AMPH (Kahlig et al., 2005). In 2012, Rodriguez- Menchaca et al. reported that AMPH-induced currents via DAT persisted even after removal of extracellular AMPH. This “persistent” current could not be observed for

NQ the physiological substrate DA or R(-)-amphetamine, which is several-fold weaker than AMPH as a releaser at DAT. In a metaphoric manner, they hypothesized that releasers act as “molecular stents” that keep the transporter in an open channel-like conformation (Rodriguez-Menchaca et al., 2012). It is evident that releasers must exhibit properties that render them distinguishable from physiological substrates. However, beyond the direct action of drugs at the MATs, it appears that a conjunction of synergistic circumstances is required to trigger transporter-mediated release. In addition to i) specific physicochemical properties of releasers (Sandtner et al., 2014), these include the ii) ion composition found in intra- and extracellular fluids, iii) the activity of kinases and other proteins interacting with MATs, iv) the composition of the plasma membrane, v) the quarternary structure of MATs and vi) access to cytosolic monoamines (Sitte and Freissmuth, 2010, Sitte and Freissmuth, 2015).

Structurally, releasers are chemically diverse. Based on the multiplicity of the effects triggered by amphetamines, Biel and Bopp investigated the structure activity relationship of amphetamines (Biel and Bopp, 1978). They defined the substantial features of amphetamine as i) an unsubstituted phenylring, ii) a two-carbon sidechain that tethers the nitrogen to the phenylring, iii) an α-methyl group and iv) a primary amine. As outlined in their original publication, modification of amphetamine might emphasize or mitigate some of its biological effects – or give rise to new activities (Biel and Bopp, 1978). Consequently, the myriad of modifications results in an overwhelming variety of amphetamine-like substances, whereas not all of them strictly fulfill the criteria to be classified as amphetamine according to Biel and Bopp, 1978 (see Figure 4).

NR

Figure 4: Chemical diversity of amphetamines. a) Definition of amphetamine (Biel Bopp, 1978): 1) an unsubstituted phenylring, 2) a two-carbon sidechain that tethers the nitrogen to the phenylring, 3) an α-methyl group and 4) a primary amine. b) to d) depict naturally occurring amphetamines. e) AMPH obeys the rules shown in a) whereas the substances shown in f) to i) do not fulfill the criteria shown in a). This figure has been taken from (Sitte and Freissmuth, 2015).

However, to exert psychoactive effects, a drug has to reach its targets within the CNS. Lipid solubility and size determine the probability of a substance to cross the BBB (van Bree et al., 1988, Waterhouse, 2003) and the ability to cross biological membranes appears to be a crucial factor that distinguishes releasers from physiological substrates (Sandtner et al., 2014). Numerous publications highlight the essential role of ions in MAT-mediated . A reduction in extracellular sodium and chloride (Pifl et al., 1995, Pifl et al.,

NS 1997) as well as an increase in extracellular potassium (Scholze et al., 2002) promotes transporter-mediated efflux. Once transported into the cytosol, lipophilic releasers may diffuse back across the membrane and are then transported back into the cell a second time and may undergo this cycle in a repetitive manner (Sandtner et al., 2014). Consequently, the increase in intracellular sodium improves the likelihood for transporter-mediated reverse transport (Sitte and Freissmuth, 2010). An important factor in reverse transport stems from proteins that interact with MATs. MATs contain various consensus sites for protein kinases (Kristensen et al., 2011, Bermingham and Blakely, 2016) and are amenable to phosphorylation (Foster et al., 2002). It has been demonstrated that amphetamines can activate kinases (Giambalvo, 1992) and pharmacological inhibition (Kantor and Gnegy, 1998, Gnegy, 2003, Seidel et al., 2005) or genetic abrogation (Chen et al., 2009) of protein kinase PKCβ attenuate amphetamine-induced reverse transport. Similar findings have been reported for CaMKIIα (Fog et al., 2006, Steinkellner et al., 2012, Pizzo et al., 2014, Steinkellner et al., 2015). Various additional kinases have been reported to interact with MATs, including PKG (Sorensen et al., 2014, Bermingham and Blakely, 2016). However, their exact role in reverse transport remains elusive. Furthermore, there is ample evidence that the lipid composition of the plasma membrane could influence reverse transport. For instance, a role of membrane- cholesterol in regulating MAT function has been suggested (Scanlon et al., 2001, Hong and Amara, 2010, Jones et al., 2012). This claim is strengthened by a crystal structure of Drosophila melanogaster DAT bound to cholesterol (Penmatsa et al., 2013). On a functional basis, depletion of membrane cholesterol markedly attenuated amphetamine-induced DA-efflux via DAT (Jones et al., 2012). Nevertheless, this observation could also be attributed to an overall reduction in DAT-activity when cholesterol is depleted (Scanlon et al., 2001, Jones et al., 2012). In an elegant study by Buchmayer et al. it was shown for the first time that amphetamines are contingent upon the presence of phosphatidylinositol-4,5-bisphosphate (PIP2) to exert full- fledged reverse transport in vitro (Buchmayer et al., 2013). Most recently, in vivo evidence has been reported that supports the conjecture that PIP2 is implicated in MAT-driven reverse transport. Mutations that perturb the interaction between DAT and PIP2 attenuated AMPH-induced locomotion in Drosophila melanogaster without affecting basal locomotion (Hamilton et al., 2014).

NT On a molecular level, SLC6 transporters exist as oligomers in living cells (Schmid et al., 2001, Kristensen et al., 2011, Anderluh et al., 2014). The hypothesis that the oligomeric state of MATs essentially affects carrier-mediated efflux has been implemented in the “oligomer-based counter-transport model “ (Seidel et al., 2005, Sitte and Freissmuth, 2015). In this model, one MAT-moiety carries the releaser into the cytosol and simultaneously, a second moiety is primed to promote efflux of the cognate monoamine (Seidel et al., 2005, Sucic et al., 2010). Increasing concentrations of releasers result in bell-shaped concentration-response curves (Pifl et al., 1999, Seidel et al., 2005, Gobbi et al., 2008, Sucic et al., 2010, Sandtner et al., 2014). Hence, efflux increases and decreases with escalating concentrations of test- drugs. Markedly, at SERT, the maximum release is reached at a concentration of the releaser that roughly reflects its Ki value (Seidel et al., 2005). The oligomer-based counter-transport model has been put forth to account for the bell-shaped concentration-response curve. It states that releasers bound to one moiety trigger a channel-like conformation of an adjacent moiety, thus enabling efflux. Concentrations above the Ki occupy both moieties, thus, preventing reverse transport and giving rise to the descending limb of the concentration-response curve (Seidel et al., 2005, Sitte and Freissmuth, 2010). In contrast, the model proposed by Sandtner et al. explains the bell-shaped curve of SERT-mediated currents by taking the lipid solubility of releasers into account. Considerable passive diffusion allows for releasers to build up in the cytosol. High concentrations of intracellular releasers affect the conformational equilibrium and promote a non-conducting state of the transporter (Sandtner et al., 2014). This may prevent monoamines engaging with their respective transporters. Nevertheless, it is worth mentioning that the concentrations of releasers that are applied in in vitro studies (up to 100 µM) exceed the concentrations that have been reported in the nervous system (Clausing et al., 1995, Zombeck et al., 2009, Siciliano et al., 2014).

No definite model for the action of releasers at plasmalemmal MATs is available at present. Yet, on common grounds, all models strictly depend on the availability of cytosolic monoamines. Under physiological conditions, cytosolic monoamines are scarce (Fon et al., 1997, Mosharov et al., 2003). By means of VMATs, cytosolic monoamines are rapidly packed into synaptic vesicles (Lin et al., 2011).

OK There is ample support for the concept that releasers affect the vesicular monoamine pools (Sulzer et al., 2005). For instance, AMPH induced-release of pre-loaded DA was higher in transfected cells that expressed both VMAT2 and DAT than in cells solely expressing DAT (Pifl et al., 1995). Notably, in the study published by Pifl et al., AMPH-evoked release could not be observed in cells devoid of DAT. However, in cells expressing DAT, release triggered by AMPH quickly returned to baseline, whereas a persistent release could be observed for cells expressing both transporters. Hence, the data strongly suggest a dual activity of AMPH at DAT and the VMATs (Pifl et al., 1995). Further support for the notion that releasers target plasmalemmal and vesicular MATs is derived from studies showing that acute pre- treatment with the VMAT2-inhibitor reserpine mitigates the effects of monoamine- releasing agents in vivo. A novel report from the group of Jonathan Javitch showed that the presence of reserpine markedly blunted AMPH and methamphetamine induced locomotion in mice. On the contrary, the effects of the MAT-inhibitor cocaine were not affected (Freyberg et al., 2016). These findings are in agreement with earlier studies reporting that reserpine-treatment diminishes amphetamine-like drug evoked monoamine release in vivo and ex vivo (Fitzgerald and Reid, 1993, Florin et al., 1995, Sulzer et al., 2005). Amphetamines alter VMAT-function and redistribute vesicular monoamines into the cytosol (Rudnick and Clark, 1993, Partilla et al., 2006). Consequently, amphetamines reduce the “quantal size”, i.e. the number of neurotransmitter molecules released per vesicle (Sulzer et al., 1995, Jones et al., 1998b). The mechanism by which monoamines are redistributed into the cytosol has been termed the “weak base hypothesis” (Sulzer et al., 2005). Sympathomimetics are weak bases with pkA-values around 9 (Sulzer, 2011). VMATs harness the proton gradient in secretory vesicles to concentrate their substrates (Gether et al., 2006). According to the weak base hypothesis, membrane diffusible amphetamines enter the synaptic vesicles and engage free protons, thus alkalinize the luminal pH (Sulzer and Rayport, 1990). A reduction in the proton gradient deprives VMATs of their driving force and reduces quantal size (Sulzer, 2011). The weak base theorem in amphetamine-action is bolstered by the observation that virtually all substances that devastate the vesicular proton gradient – for instance the weak bases ammonium chloride and chloroquine - reduce quantal size (Sulzer and Rayport, 1990). Furthermore, amphetamines might reverse the direction of transport by VMATs. Akin to their action at the plasmalemmal MATs, amphetamines bind to the substrate-

OL binding site in VMATs (Schuldiner et al., 1993). As substrates of VMAT2 (Partilla et al., 2006), amphetamines could release monoamines into the cytosol by facilitated exchange diffusion or even decrease the proton gradient by being transported by VMATs. However, the exact mechanisms remain elusive (Sulzer, 2011). It is noteworthy to mention that amphetamines do not only elevate the cytosolic concentration of monoamines via their action at vesicular pools. Amphetamines support the formation of catecholamines by enhancing the activity of TH (Kuczenski, 1975). TH converts tyrosine into L-dihydroxyhenylalanine (L-DOPA), hence, catalyzes the rate-limiting step in the synthesis of the catecholamines DA, NE and epinephrine (Nagatsu et al., 1964, Levitt et al., 1965, Larsen et al., 2002). Furthermore, various amphetamine-like drugs inhibit the enzymatic degradation of monoamines by acting as non-substrate competitive inhibitors of MAO-A and B (Seiden et al., 1993, Sulzer et al., 2005, Sulzer, 2011). Hence, interplay of the above- mentioned effects is believed to provide cytosolic monoamines for reverse transport via DAT, NET and SERT.

New Psychoactive Substances

An emerging phenomenon has been the introduction of new psychoactive substances (NPS) into the global drug market (Miliano et al., 2016). In practical terms, NPS provide powerful and easily accessible compounds that serve as surrogates for illicit drugs (Baumann and Volkow, 2016). NPS, also referred to as “legal highs”, “research chemicals”, “plant food” or “bath salts”, are sold over the Internet or in retail shops (Baumann et al., 2013a, Tyrkko et al., 2016). To circumvent laws that regulate the sale of psychoactive chemicals, these compounds are often labeled “not for human consumption” (Baumann and Volkow, 2016). Chemically, NPS encompass pharmaceutically designed substances that were never commercialized and used in clinical medicine, or newly synthesized substances (Brandt et al., 2014). The latter can be based on existing and regulated drugs with psychoactive properties (Lewin et al., 2014). In that case, chemists extract knowledge from the scientific and patent literature about the structures of regulated compounds that target transporters and receptors in the nervous system. The subsequent elaborate modifications of the parental drugs can result in powerful and legal psychoactive substances (Baumann et al., 2014b, Lewin et al., 2014).

OM Strikingly, NPS appear on the drug market at astonishing rates –approximately 100 new substances per year (Meyer, 2016). NPS range from synthetic (“spice”) that elicit cannabis-like effects to psychostimulants that mimic the action of MDMA and cocaine (Baumann et al., 2014b, Miliano et al., 2016, Soussan and Kjellgren, 2016). The problem of new psychoactive substances (NPS) has long been ignored, which provided fertile grounds for some substances to become well-established members of the drug market (Sitte and Freissmuth, 2015) (European Monitoring Centre for Drugs and Addiction, 2015). Misleadingly, NPS are distributed under innocuous names, e.g. plant food (Kiyatkin et al., 2015), which might even lead to the false assumption that some NPS are safe (Baumann and Volkow, 2016). However, NPS reveal strikingly potent activity at pharmacologically relevant targets. For instance, “spice” products contained synthetic cannabinoids, such as JWH-018 and JWH-073, which imitate the effect of Δ9-tetrahydrocannabinol (THC). Remarkably, the active ingredients of “spice” revealed affinities towards the cannabinoid receptors (CB) CB1 and CB2 that were greater than - or at least comparable to- those observed for THC (Lindigkeit et al., 2009). Other prominent examples for NPS that received attention in the media are the “bath salts" 3,4-methylendioxypyrovalerone (MDPV, “super coke”), 3,4- methylenedioxymethcathinone () and 4-methylmethcathinone (mephedrone, “M-CAT”, “meow meow”), which exert powerful stimulatory effects (Baumann et al., 2013a). MDPV, mephedrone and methylone are cathinone derivatives. On a molecular level, mephedrone and methylone act as substrates at MATs and trigger full-fledged release of monoamines via MATs (Baumann et al., 2012). In contrast, MDPV potently inhibits DAT and NET (Baumann et al., 2013b). Fascinatingly, MDPV shows only moderate affinity towards SERT, but is several-fold more potent than cocaine at DAT and NET (Baumann et al., 2013b). Aside from their rewarding and stimulating properties, the bath salts MDPV, mephedrone and methylone have been associated with adverse side effects, including hyperthermia, and even death (Prosser and Nelson, 2012, Baumann et al., 2013a). Consequently, many countries installed laws to render the “first generation ” illegal (Schifano et al., 2011, Baumann et al., 2013a). Soon after the legislative ban on mephedrone, MDPV and methylone, the drug market was inundated with “second-generation cathinone” analogs, i.e. 4-methyl-N- ethylcathinone (4-MEC) and 4-methyl-α-pyrrolidinopropiophenone (4-MePPP) (Saha

ON et al., 2015). This exemplifies the elastic and adaptable nature of the NPS market. From a chemical perspective, 4-MEC and 4-MePPP closely resemble mephedrone. However, the chemical modification drastically affects the pharmacological profile. Rather than acting as transportable substrate, 4-MePPP acts as a non-transportable inhibitor at MATs. 4-MEC displays “hybrid activity” as substrate of SERT and as blocker at DAT (Saha et al., 2015). As already described by Biel and Bopp, chemical modification may change the activity in relation to the parental compound (Biel, Bopp). Therefore, the overwhelming amount of NPS is associated with rich pharmacology (Sitte and Freissmuth, 2015), as displayed by mephedrone, 4-MEC and 4-MePPP. In general, NPS enter the drug market without knowledge of their and pharmacokinetics (Meyer, 2016). Hence, it is an important task to unravel the molecular mechanisms by which individual NPS exert their bioactive effects, not only to estimate potential neurotoxic effects (Baumann et al., 2014a) but also for the emergency medical care units that are confronted with NPS intoxications. Further, it is crucial to investigate the metabolic fate of NPS to identify potentially toxic and psychoactive metabolites. Detailed knowledge about the pharmacodynamics and pharmacokinetics would further allow for developing forensic assays to detect NPS (Baumann and Volkow, 2016). Due to their chemical variety, detection of NPS in biological samples is a challenging task (Favretto et al., 2013). Based on the performance enhancing properties, psychostimulants carry the underlying potential for use as doping agents (Docherty, 2008) and NPS have already been detected in racehorses and athletes (Wang et al., 2012, Li et al., 2014, Kwiatkowska et al., 2015).

OO Aims of this thesis

The aims of the two embedded research articles were as follows:

i) Assessing the potential biological activity of the phase 1 metabolites of mephedrone at monoamine transporters ii) Deciphering the molecular mechanism of action of the phase 1 metabolites of mephedrone at monoamine transporters, i.e. differentiating substrate-type releasers from inhibitors iii) Elucidating whether systemically-administered phase 1 metabolites affect the behavior of test animals iv) Determining the pharmacological profile of the new psychoactive substance 3-fluoro-phenmetrazine (3-FPM) and its positional isomers 2- and 4-FPM at monoamine transporters.

Aims i)-iii) address the question whether the phase I metabolites of mephedrone exert bioactive effects at monoamine transporters. As revealed by radiotracer flux experiments, this study provides evidence that metabolic breakdown of mephedrone results in bioactive metabolites that act as releasers at monoamine transporters. Further, systemic administration of the N-demethylated metabolite, nor-mephedrone, triggers release of serotonin and dopamine in nucleus accumbens and elevated locomotion in rats, akin to mephedrone. Aim vi) relates to an experimental study that was designed to decipher the pharmacological profile of fluorinated phenmetrazines. 3-FPM is a NPS that recently appeared on drug markets. Yet, no detailed information is available on the mechanism of action of 3-FPM (McLaughlin et al., 2016). The positional isomers 2- FPM and 4-FPM were included in that study, since 2- and 4-FPM may appear in the drug markets to replace 3-FPM.

OP Materials and Methods

Declaration

I, Felix Paul Anton Mayer, declare first authorship of the presented book chapter, entitled “Application of a combined approach to identify new psychoactive street drugs and decipher their mechanisms at monoamine transporters“, which contains a detailed description of the applied methods. I declare that I wrote the first draft of this book chapter and that I received substantial input and support from Prof. Dr. Harald H. Sitte. Subsequently, together with Prof. Dr. Harald H. Sitte, I further elaborated the style and the content after receiving remarks from the editors, Dr. Michael H. Baumann and Prof. Dr. Richard A. Glennon.

Publication

Felix P. Mayer, Anton Luf, Constanze Nagy, Marion Holy, Rainer Schmid, Michael Freissmuth, and Harald H. Sitte (2016); “Application of a combined approach to identify new psychoactive street drugs and decipher their mechanisms at monoamine transporters“; accepted, “Neuropharmacology of New Psychoactive Substances (NPS): The Science Behind the Headlines”, Current Topics in Behavioral Neurosciences, a fully edited version is available online: doi:10.1007/7854_2016_63

 

OQ Application of a combined approach to identify new psychoactive street

drugs and decipher their mechanisms at monoamine transporters

Felix P. Mayer1, Anton Luf2, Constanze Nagy3, Marion Holy1, Rainer Schmid2, Michael Freissmuth1, and Harald H. Sitte 1,4*

1Medical University Vienna, Center of Physiology and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13a, A-1090 Vienna, Austria

2Clinical Department of Laboratory Medicine, Medical University of Vienna, Waehringer Guertel 10-20, 1090 Vienna, Austria

3checkit!-Suchthilfe Wien GmbH, Gumpendorfer Gürtel 8, 1060 Vienna, Austria

4Center for Addiction Research and Science - Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria

*Author to whom correspondence should be addressed:

Harald H. Sitte, Medical University of Vienna, Institute of Pharmacology, Center for Physiology and Pharmacology, Waehringer Strasse 13a, A-1090 Vienna, Austria; Tel: +43-1- 40160-31323; Fax: +43-1-40160-931300

E-mail: [email protected]

1

Abstract

Psychoactive compounds can cause acute and long-term health problems and lead to addiction. In addition to well-studied and legally controlled compounds like cocaine, new psychoactive substances (NPS) are appearing in street drug markets as replacement strategies and legal alternatives. NPS are effectively marketed as ‘designer drugs’ or

‘research chemicals’ without any knowledge of their underlying pharmacological mode of action, their potential toxicological effects and obviously devoid of any registration process.

As of 2016, the knowledge of structure-activity relationships for most NPS is scarce, and predicting detailed pharmacological activity of newly-emerging drugs is a challenging task.

Therefore, it is important to combine different approaches and employ biological test systems that are superior to mere chemical analysis in recognizing novel and potentially harmful street drugs. In the current chapter, we provide a detailed description of techniques to decipher the molecular mechanism of action of NPS that target the high affinity transporters for dopamine, norepinephrine and serotonin. In addition, this chapter provides insights into a combined approach to identify and characterize new psychoactive street drugs of unknown content in a collaboration with the Austrian prevention-project ‘checkit!’.

Key words

New psychoactive substances, research chemicals, monoamine transporters, psychostimulants, amphetamine, cocaine, dopamine, serotonin, norepinephrine, bath salts, analytical identification

2

1 Introduction ...... 4 2 Methods for Assessing Drug Actions at Transporters ...... 8 2.1 HEK293 cell culture ...... 8 2.2 Uptake inhibition assays ...... 10 2.3 Release assays...... 11 3 Interpreting Data from Transporter Assays ...... 13 3.1 Uptake inhibition assays ...... 13 3.2 Release assays...... 16 4 Biological assays to identify street drugs of unknown content ...... 22 5 Choosing the appropriate expression system ...... 24 6 Discussion ...... 25 References ...... 28

3

1 Introduction An emerging problem in recent years is the increasing abuse of new psychoactive substances (NPS) – so called “legal highs”, “bath salts” or “research chemicals” available on street drug markets. NPS are mostly distributed over the Internet and comprise failed pharmaceuticals, like the bath salt 3,4-methylenedioxypyrovalerone (MDPV, “cloud nine”)

(Brandt, King & Evans-Brown, 2014), or synthetic substances of novel structure. The latter are based on known chemical structures that target receptors or transporters for neurotransmitters in the nervous system (Lewin, Seltzman, Carroll, Mascarella & Reddy,

2014). However, due to chemical modification of the parent drugs, NPS evade current drug control legislation (Brandt, King & Evans-Brown, 2014). A major challenge associated with

NPS is the flexibility of vendors to adapt to changes within legislative boundaries. For instance, JWH-018, a former unregulated cannabinoid receptor , was found to be an active ingredient of the “legal high” product “Spice”. Only four weeks after the legislative ban on JWH-018, the unregulated analogue JWH-073 began appearing in “Spice” preparations (Baumeister, Tojo & Tracy, 2015; Lindigkeit et al., 2009). The replacement of

JWH-018 with JWH-073 represents a perfect example of the often-cited “cat-and-mouse- game” (Baumeister, Tojo & Tracy, 2015; Brandt, King & Evans-Brown, 2014) whereby the ban of a given substance inevitably results in the appearance of novel and uncontrolled substances as a replacement strategy. Strikingly, the number of NPS (in total: 251 in 2012) has already overtaken the number of controlled substances (in total: 234) (World drug report 2013). The alarming increase in NPS available on recreational drug markets prompted the United Nations Office on Drug and Crime (UNODC) to launch an early warning advisory

4 system on NPS in 2013 (UNODC, 2013) to provide a variety of up-to-date information on an international scale.

Unfortunately, knowledge about the pharmacology of most NPS is limited, if not missing altogether (Baumann, Solis, Watterson, Marusich, Fantegrossi & Wiley, 2014).

Moreover, high-dose or chronic exposure to NPS may result in severe medical conditions, including , tachycardia and even death (Baumann, Solis, Watterson, Marusich,

Fantegrossi & Wiley, 2014; Miliano, Serpelloni, Rimondo, Mereu, Marti & De Luca, 2016;

Prosser & Nelson, 2012), (Ross, Watson & Goldberger, 2011). NPS satisfy a broad spectrum of individual demands for recreational drugs (Soussan & Kjellgren, 2016), a spectrum which ranges from legal alternatives for cannabinoids and hallucinogens to stimulants.

Stimulant-like NPS exert amphetamine- or cocaine-like pharmacological effects by interfering with monoaminergic signalling pathways (Baumann, Solis, Watterson, Marusich,

Fantegrossi & Wiley, 2014; Miliano, Serpelloni, Rimondo, Mereu, Marti & De Luca, 2016).

Simplified, psychostimulants come in two flavours as: i) cocaine-like uptake inhibitors or ii) amphetamine-like releasers. The first mentioned class, including cocaine and , exerts its effects by inhibiting the high-affinity monoamine transporters

(MATs) for dopamine (dopamine transporter, DAT), norepinephrine (norepinephrine transporter, NET) and serotonin (serotonin transporter, SERT). As a consequence, the monoamine concentration increases in the synaptic cleft and activates pre- and postsynaptic receptors (Torres, Gainetdinov & Caron, 2003). The second-mentioned class, including amphetamine and its analogues like 3,4-methylenedioxymethamphetamine (MDMA,

“ecstasy”), triggers a transporter-mediated reverse transport of monoamines from the cytoplasm into the extracellular space (Sitte & Freissmuth, 2010). It is currently believed that releasers are transported as substrates by DAT, NET and SERT and reverse the normal

5 direction of transport flux (Sitte & Freissmuth, 2015). Consequently, releasers elevate the extracellular concentration of monoamines independently from vesicular release events

(Sulzer, Sonders, Poulsen & Galli, 2005).

Nevertheless, MATs at the plasma membrane operate in concert with vesicular monoamine transporters (VMATs) to refill the vesicular storage pools (Egana et al., 2009). In addition to their actions at MATs, amphetamine-like drugs (releasers) have been shown to interact with VMATs (Schuldiner, Steiner-Mordoch, Yelin, Wall & Rudnick, 1993) and to release monoamines from synaptic vesicles into the cytosol (Sulzer, Sonders, Poulsen & Galli,

2005). The concomitant availability of monoamines for reverse transport, and the inverted direction of flux for plasmalemmal MATs, has been hypothesized to be crucial for the actions of amphetamine-like drugs (Freyberg et al., 2016; Sitte & Freissmuth, 2015). Most importantly, a link between amphetamine-like drugs and neurotoxicity has been established

(Baumann et al., 2014; Pifl, Reither & Hornykiewicz, 2015). Hence, to assess the potential risk of psychostimulant NPS on neuronal function and overall health status, it is imperative to decipher their molecular mode of drug action at MATs.

Structure-activity relationships (SAR) studies have evolved from providing useful representations of docked molecules to – in many cases - promising tools to shape and form our understanding of the molecular mechanism of action of drugs, as these studies shed light onto the molecular determinants of these actions . Certainly, this evolution has been triggered by the development of more reliable (and biochemically verified) homology models (Stockner et al., 2013) and the availability of more and more structures of bacterial transporter homologs (Penmatsa & Gouaux, 2014; Yamashita, Singh, Kawate, Jin & Gouaux,

2005) and even structures derived from drosophila (Penmatsa, Wang & Gouaux, 2013) and human species (Coleman, Green & Gouaux, 2016). However, as of 2016, only a few SAR

6 studies on psychostimulant NPS exist (Bonano et al., 2015; Kolanos, Sakloth, Jain, Partilla,

Baumann & Glennon, 2015; Saha et al., 2015; Sandtner et al., 2016). Future studies should be based and designed on the current state-of-the-art techniques and approaches to enhance our understanding of drug actions at MATs. This could certainly also help to bypass the tedious and time-consuming intermediate step of biological evaluation of each of the compounds found on the illicit drug market.

As a result of the current dynamics of the drug markets, society is flooded with a variety of modified substances. Previous studies reveal that chemical modifications of psychostimulants might switch their activity from amphetamine-like to cocaine-like or

MDMA-like drugs (Saha et al., 2015). Consequently, the structural modifications of known substances may result in ineffective or even toxic substances. A major threat for the physical and mental health of drug users is that NPS are rarely sold under their real name or in their pure form on the street.

To identify potential harmful substances, i.e. adulterants or drug combinations, the

City of Vienna in Austria launched the prevention project known as ‘checkit!’. Without the risk of criminalization, drug users can have their drugs anonymously tested for active ingredients and adulterants. In addition to a permanent location based in Vienna, ‘checkit!’ offers “on site” analysis (Brunt et al., 2016). To reach out to people at rave parties and music festivals, a mobile laboratory has been installed in a bus. This unique project not only reduces the occurrence of severe intoxications and offers harm reduction information, but also provides valuable insights into the current street drug market situation and allows for documenting market entries of NPS. The drug user provides a few milligrams of his/her purchased product, which is then analysed by high performance liquid chromatography coupled with mass-spectrometry (HPLC-MS). Immediately after analysis by ‘checkit!’, the

7 user receives information on the content of the drug sample. If the analysis yields inconclusive results, the content is classified as “unknown”(Ostermann et al., 2014). A combined approach of high resolution mass spectrometry (HRMS) and biological activity assays performed in heterologous expression systems described herein can be used to identify the content of “unknown” test drugs and the underlying pharmacological activity profile (Rosenauer, Luf, Holy, Freissmuth, Schmid & Sitte, 2013). On a monthly basis,

‘checkit!’ publishes detailed warning lists, which are also forwarded to the European Drug

Monitoring Centre for Drugs and Drug Addiction (EMCDDA) in Lisbon, Portugal. These lists contain information on high-dose drug preparations or adulterated and combined drug mixtures that should be handled with special care.

Recently, collaboration between the Sitte research group and the ‘checkit!’ program has shed light on the reason for the ubiquitous use of levamisole as adulterant in cocaine. In addition to its bitter taste, a metabolite of levamisole, , exerts amphetamine-like effects at MATs (Hofmaier et al., 2014). In the current chapter, we discuss techniques that have been established and successfully applied to identify substances that target MATs and experimental approaches to discriminate amphetamine-like drugs from non-transported inhibitors. Furthermore, we discuss the application of the described techniques to identify street drugs of unknown content.

2 Methods for Assessing Drug Actions at Transporters

2.1 HEK293 cell culture Human embryonic kidney cells (HEK293) are cultured in 10-cm cell culture dishes

(Sarstedt, Germany) in Dulbecco’s Modified Eagles Medium (DMEM; high glucose 4500

8 mg/L), sodium bicarbonate and L-glutamine (Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS). To maintain the cells in a sub-confluent state, the cells are washed with phosphate buffered saline (PBS) every 4 days and exposed to 1 mL of trypsin for 2-3 minutes at room temperature. After establishing a monocellular suspension by trituration, 0.7-1.0*106 cells are transferred into a new 10-cm dish and DMEM supplemented with FBS is added to a final volume of 10 mL. In the case of HEK293 cells stably expressing the desired MATs, the selection process is maintained by adding the appropriate antibiotics according to the protocol of the vector supplying company. If needed, the cell culture medium may be further supplemented with penicillin (100 IU/100 mL) and streptomycin (100 μg/100 mL).

Transfection of HEK293 cells with the MAT of interest can be achieved by application of the CaPO4 method (Chen & Okayama, 1987), which is reliable and inexpensive. Prior to transfection, the cells should reach a confluency of 35 to 45%. This can be achieved by seeding 1.8*106 cells into a 10-cm dish 24 hours prior to transfection. For each 10-cm dish, mix 20 μL of DNA (1 μg/μL) with 480 μL of 0.26 M CaCl2 in H2O. Subsequently, transfer the

DNA containing mixture into 500 μL of Hepes-buffered saline (HEBS) and let the mixture sit for 6 minutes at room temperature. After 6 minutes, a fine-grained DNA-Ca2+ precipitate should be visible. Add the solution with the DNA-precipitate dropwise onto the cells. Let cells sit for 3.5-6 hours at 37°C, with exposure to 5% CO2. Afterwards, aspirate the transfection medium and add 1 mL of glycerol (10 vol%) to the cells and remove it immediately. Wash the cells with 7.5 mL of PBS and add 10 mL of DMEM, supplemented with FBS. If MAT-cDNAs are used that carry a fluorescent protein tag such as green fluorescent protein (GFP) or any other fluorescent protein, expression can be monitored the next day by use of a fluorescence microscope. If the transfected MATs do not carry a tag

9 allowing visual assessment of expression, perform a single point uptake of tritiated substrate in absence and presence of specific inhibitors to verify correct MAT expression at the plasma membrane.

To generate cell lines stably expressing the MAT of choice, add the antibiotic listed in the datasheet of the applied expression vector 48 hours after transfection. Maintain a high- selection pressure for up to 5 days until only viable single cells are present. To establish monoclonal cell lines, pick 1 to 10 individual cells with a sterile 200 μL pipette tip. Expand the clones in individual cell culture dishes and repeat this step 2-3 times. Finally, test each individual clone for its transport characteristics, i.e. KM and Vmax for reference substrates

(endogenous MAT substrates or MPP+) (Hilber et al., 2005). For the uptake inhibition experiments, monocellular suspensions of HEK293 cells expressing the desired MAT are seeded at 40000 cells per well onto poly-D- coated 96-well plates (Sarstedt) in a final volume of 200 μL/well 24 hours prior to the experiment. For release experiments, poly-D- lysine coated glass coverslips (5 mm in diameter) are placed into 96-well plates.

Subsequently, 40000 cells per well are seeded onto the glass coverslips in a final volume of

200 μL/well 24 hours prior to the experiment.

2.2 Uptake inhibition assays Sodium bicarbonate buffer containing DMEM is removed from the cells and replaced with Krebs-HEPES-buffer (KHB, 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, and

1.2 mM MgSO4, 5 mM D-glucose, pH adjusted to 7.3 with NaOH) at a final volume of 200

μL/well. Subsequently, cells are exposed to KHB containing various concentrations of test drug for 5 minutes to achieve equilibration. Afterwards, tritiated substrate ([3H]-MPP+ at a final concentration of 20 nM for DAT and NET or [3H]-5-HT at a final concentration of 100 nM

10 for SERT) is added. The uptake incubation is terminated after 180 (DAT and NET) or 60 seconds (SERT) by removing the tritiated substrate and washing the cells with 200 μL of ice- cold KHB. Finally, the cells are lysed with 1% SDS (100 μL/well) and transferred into counting vials filled with 2 mL of scintillation cocktail. Uptake of tritiated substrate is determined with a beta-scintillation counter. Non-specific uptake via DAT, NET or SERT is assessed in presence of 10 μM mazindol, or paroxetine, respectively, and subtracted from all data to yield specific uptake. Uptake in the absence of test drugs is defined as 100% and uptake in the presence of drugs is expressed as a percentage of control uptake. The half maximal inhibitory concentration is determined by non-linear regression fits according to the equation: [Y=Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))]

2.3 Release assays

Dynamic Superfusion Assay

MAT-mediated reverse transport is assessed by use of a dynamic superfusion apparatus as described in detail elsewhere (Pifl, Drobny, Reither, Hornykiewicz & Singer,

1995). In brief, MAT-expressing cells grown on 5-mm glass coverslips are exposed to 0.1 μM

[3H]-MPP+ (DAT and NET) or 0.4 μM [3H]-5-HT (SERT) at 37°C for 20 minutes. Subsequently, the cells are transferred into 12 individual small cylindrical chambers (8 mm in diameter; volume 200 μL) and superfused with KHB at a flow rate of 0.7 mL/min for 40 minutes to establish a stable basal release of tritiated substrate. To ensure that the experiment is conducted at a constant temperature, the tubes delivering KHB to the individual chambers are placed into a water bath set to 25°C. After a 40-minute wash-out phase, three two-

11 minute fractions are collected which represent the untreated (i.e., basal) release.

Afterwards, the cells are exposed to monensin (10 μM) or vehicle for four fractions, prior to the addition of test drugs in the presence or absence of monensin for five fractions (the reason for the addition of monensin will be discussed in detail below). Finally, the cells are superfused with 1% SDS for another three fractions and total radioactivity present in each fraction is determined by a beta-scintillation counter (Perkin Elmer, Waltham, MA, USA). For analysis, the release of pre-loaded substrate is expressed as fractional rate, i.e. the amount of released radioactivity per fraction is expressed as percentage of total radioactivity present in the cells at the beginning of that fraction (Sitte, Scholze, Schloss, Pifl & Singer, 2000).

Static Batch Release Assay

Originally described by Rudnick and co-workers for MAT-expressing cells in 1995, the static batch release assay serves as a technique to identify substrate-induced efflux (Wall, Gu

& Rudnick, 1995). HEK293 cells expressing the MAT of interest are preloaded with tritiated substrate (0.05 μM in KHB, 100 μL/well) for 20 minutes (37°C). Subsequently, the cells are washed three times with KHB (200 μL/well) at room temperature to rinse away any tritiated substrate free in solution, which improves the signal-to-noise ratio. Finally, the test drugs (in

KHB, 100 μL/well) are added at a concentration that inhibits uptake via the respective MAT by 50%, and test drugs are always compared to an established MAT substrate, e.g.

(+)amphetamine. To determine the specificity of drug-induced reverse transport, controls are performed in the presence of selective MAT-inhibitors; e.g. 10 μM of mazindol for DAT and NET and 10 μM of paroxetine for SERT. After ten minutes, the supernatant is transferred into liquid scintillation counting vials. The cells are lysed in 100 μL of 1% SDS and transferred into independent counting vials. Total radioactivity present in the supernatant and the cell

12 lysate is set as 100%, and the amount of [3H]-substrate present in the supernatant is expressed as percentage of the total.

3 Interpreting Data from Transporter Assays

3.1 Uptake inhibition assays Drugs that target MATs inhibit the uptake of their cognate neurotransmitter substrates in a dose-dependent manner (Figure 1). The apparent IC50 values may vary with expression levels and the cell system used (see below, section 5). Hence, cocaine may be applied as an internal reference drug for comparison to the potencies of the test compounds under scrutiny. Figure 1 depicts the effects of cocaine and MDPV on DAT and SERT mediated uptake in HEK293 cells. As described previously (Baumann et al., 2013), MDPV inhibits DAT- mediated uptake with much higher potency than cocaine (IC50 values of 0.015 μM vs. 0.36

μM for MDPV and cocaine, respectively). On the contrary, MDPV is strikingly less potent than cocaine as an inhibitor at SERT (IC50 ~6 μM for MDPV as compared to 0.22 μM for cocaine). As a negative control, one might examine the effects of test drugs at the GABA- transporter (Rosenauer, Luf, Holy, Freissmuth, Schmid & Sitte, 2013); this transporter should be essentially unaffected by any given MAT substrate or inhibitor.

13

Figure 1: Effects of MDPV and cocaine on DAT- and SERT-mediated uptake in HEK293 cells.

The human isoforms of DAT (left) and SERT (right) were stably expressed in HEK293 cells, and incubated with increasing concentrations of cocaine or MDPV. Uptake of [3H]-MPP+ by DAT or [3H]-5-HT by SERT is expressed as a percentage of uptake in the absence of inhibitors.

Nonspecific uptake is determined in the presence of 10 μM mazindol or paroxetine for DAT or

SERT, respectively.

Uptake inhibition assays allow for the identification of drugs that counteract MAT- mediated uptake. However, this assay lacks the ability to differentiate amphetamine-like substrates from non-transported inhibitors (Baumann et al., 2012; Baumann, Partilla &

Lehner, 2013). Aside from triggering reverse transport, amphetamine-like drugs, i.e., drugs that act as “releasers” similar to (+)amphetamine, bind to the orthosteric site on transporters and are subsequently transported as substrates. However, it is worth mentioning that some drugs that share structural features with (+)amphetamine do not trigger transporter-mediated efflux and act as non-transported inhibitors, with methylphenidate being the most prominent example (Sitte & Freissmuth, 2015). Hence,

14 drugs acting as transporter substrates compete for the orthosteric binding sites with the natural neurotransmitter substrates, and engender dose-dependent reductions in uptake of tritiated substrate. As shown in Figure 2, the former legal high and MAT-substrate mephedrone (Baumann et al., 2012; Baumann, Partilla & Lehner, 2013) inhibits DAT- mediated uptake with increasing concentrations.

Figure 2: Effects of mephedrone and cocaine on DAT-mediated uptake in HEK293 cells.

DAT-expressing HEK293 cells were incubated with the indicated concentrations of cocaine or mephedrone (MEPH). Uptake of [3H]-MPP+ is expressed as per cent of uptake without inhibitors present, and nonspecific uptake is defined in the presence of mazindol (10 μM).

The non-transported inhibitor MDPV and the MAT-substrate mephedrone share a similar profile of activity in uptake inhibition assays. This illustrates that assays dedicated to reveal drug-induced reverse transport are needed to identify the precise mode of action for drugs

15 which target transporters. Nevertheless, uptake inhibition assays provide a fast and reliable tool to identify the compounds that interact with MATs.

3.2 Release assays The major advantage of the dynamic superfusion experiments is the elimination of back-and-forth movements of substrates by diffusion (Scholze, Zwach, Kattinger, Pifl, Singer

& Sitte, 2000). The constant flow rate ensures that released substrates are cleared from the cellular vicinity. This prevents transporter-mediated reuptake or diffusion events that might counteract reverse transport induced by test drugs. Additionally, and contrary to static release assays, the temporal resolution of superfusion enables deciphering the time-course of effects for each drug. Monoamine transporters of the SLC6 family utilize the pre-existing sodium gradient across cell membranes as a driving force (Rudnick & Clark, 1993).

Application of the selective Na+/H+ ionophore monensin (Mollenhauer, Morre & Rowe,

1990) dissipates the sodium gradient by allowing sodium entry into the cytosol. As a result, the increase in intracellular sodium fosters MAT-mediated reverse transport and selectively augments substrate induced reverse transport. As a consequence, only efflux triggered by

"true" substrates is sensitive to enhancement by monensin, while the effects of non- transported inhibitors remain unaffected (Sandtner et al., 2016). A representative experiment showing the effect of monensin on transporter-mediated efflux is given in Figure

3. The addition of the MAT-substrate para-chloroamphetamine (PCA, 3 μM) robustly elevates the basal release of tritiated substrate via SERT. The presence of monensin (10 μM) further augments PCA-triggered reverse transport. In striking contrast, application of the

SERT-inhibitor paroxetine (3 μM) does not result in tritium outflow, and no difference between paroxetine plus monensin or vehicle can be observed.

16

Figure 3: The impact of monensin on SERT-mediated reverse transport in HEK293 cells.

HEK293 cells stably expressing human SERT were preloaded with [3H]-MPP+ and superfused.

Pretreatment with monensin (MON, 10 μM) augments the reverse transport triggered by para-chloramphetamine (PCA, 3 μm) versus the vehicle pretreated condition. Application of the SERT-inhibitor paroxetine (PAROX, 3 μM) does not result in elevated [3H]-MPP+-outflow and is insensitive to pretreatment with monensin. The presence of monensin, vehicle, PCA and PAROX is indicated by black boxes.

17

As exemplified in Figure 3, only the effects of transporter substrates are sensitive to the presence of monensin. This property precludes the misinterpretation of “pseudo”-efflux events. Previous studies have shown that MAT-inhibitors can unmask a basal loss of tritiated substrate from the preloaded cells (Sitte, Scholze, Schloss, Pifl & Singer, 2000). Despite the advantages of superfusion methods, limited yet measurable amounts of tritiated substrate can leak from cells via simple diffusion. This leak process is normally counteracted by the activity of MATs in transfected cells. However, in the presence of MAT inhibitors, the re- uptake of extracellular substrates is precluded. Thus, the presence of MAT inhibitors can elevate the amount of tritiated substrate in the superfusate and might be interpreted as drug-induced efflux. As shown in Figure 4a, by magnification of the Y-axis, the addition of

MDPV elevates the basal release of [3H]-MPP+ from HEK293 cells stably expressing human

DAT. Considered in isolation, it might be interpreted that MDPV acts as a weak amphetamine-like substrate, although the effect is rather modest and does not reach statistical significance. By contrast, when compared to the effects of PCA in Figure 3, the presence of monensin reveals that MDPV does not induce reverse-transport via the sodium- dependent DAT (Figure 4b), which is in line with previous studies that conclusively substantiated that MDPV acts as non-transported inhibitor at DAT (Baumann et al., 2013).

This example highlights that observing the effects of test drugs in the absence and the presence of monensin is a direct and effective strategy to distinguish between drugs which act as MAT substrates versus those which act as non-transported inhibitors.

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Figure 4: Effects of MDPV in the absence and presence of monensin on DAT mediated reverse transport in HEK293 cells. HEK293 cells stably expressing human DAT were pre- loaded with [3H]-MPP+ and superfused. (A) Addition of MDPV slightly elevates the basal release of tritiated substrate. (B) The presence of monensin (MON) does not augment the modest effect of MDPV. The addition of substances is indicated by black bars and arrows.

However, it is important to stress potential pitfalls associated with the use of monensin. As a Na+/H+-ionophore, monensin dissipates sodium gradients but also alters the intracellular pH. Monoamine neurotransmitters (i.e., dopamine, norepinephrine and 5-HT) are weak bases with pKa values greater than 8. Consequently, alterations in the intracellular concentration of H+ ions can affect the ratio between protonated and unprotonated forms of these amines. In 2000, Scholze and coworkers demonstrated that the addition of monensin alone elevated the amount of tritium in superfusates when [3H]-5-HT was used to preload

HEK293 cells in the absence of any plasmalemmal transporter expression (Scholze, Zwach,

Kattinger, Pifl, Singer & Sitte, 2000). The increase in preloading time up to one hour was enough to efficiently load the cells with tritiated 5-HT by a non-transporter-related mechanism. A likely explanation for this observation is that elevation of intracellular pH by 19 monensin increases the unprotonated and more lipophilic form of 5-HT, and facilitates diffusion of 5-HT across cellular membranes (Rudnick, Kirk, Fishkes & Schuldiner, 1989).

Regarding the "pseudoefflux" described by Scholze et al. (2000) and Sitte et al. (2000), the elevation of tritiated substrates in the superfusate by MAT-blockers could be misinterpreted as the effect of an amphetamine-type releasing agent, as outlined above. If the results obtained with [3H]-5-HT are unclear, the use of [3H]-MPP+ is an effective countermeasure.

The permanent charge of [3H]-MPP+ prevents passive diffusion across cellular membranes to a large extent. Therefore, MPP+ reveals only transporter-mediated translocation events.

It is also noteworthy that transporter-mediated release induced by amphetamine-like substrates results in a bell-shaped dose-response curve. As extensively studied by Seidel and colleagues (Seidel et al., 2005), high concentrations of test drugs may counteract transporter-mediated reverse transport, if the applied concentration of the drug of interest exceeds the determined IC50 by several-fold. Therefore, uptake inhibition assays not only serve as a tool to identify potential candidate drugs for further analysis but also provide the basis to identify the correct concentration range for superfusion studies, i.e. the half- maximal inhibitory concentration for MAT-mediated uptake.

A major disadvantage of dynamic superfusion experiments is that large amounts of buffer and substance are required. To bypass this obstacle - if only small amounts of test drugs are available, two possibilities exist: i) electrophysiological investigations, which are well suited to discriminate substrates from inhibitors (Sandtner et al., 2016); the electrogenic uptake process mediated by MATs clearly identifies substrates based on drug- induced inward sodium currents while non-transported inhibitors fail to induce such currents; ii) an alternative assay that requires only minute amounts of the drug under scrutiny - the static batch release assay. Analogous to the dynamic superfusion experiments,

20 the experimental strategy in the static batch assay is based on the fact that MAT-mediated transport can run in reverse. Hence, MAT-expressing cells are preloaded with tritiated substrate and incubated with buffer containing the drug of interest. Subsequently, the amount of radioactivity present in the incubation buffer is determined and expressed as percentage of total radioactivity present (i.e., in cells and in buffer), as determined by disintegrating the cells with 1% SDS. The effect of test drugs needs to be determined in the presence and absence of specific MAT-inhibitors. Specific MAT inhibitors markedly reduce the amount of tritium in the supernatant if the cells are incubated with substrate-type drugs

(Figure 5). On the contrary, the effects of drugs that act as non-transported inhibitors are insensitive to the presence of other MAT inhibitors. Even though the dynamic superfusion assay yields results of higher quality, the static batch assay provides a reliable readout to identify substrate-type drugs targeting MATs.

Another limitation of dynamic superfusion systems is that determining the half- maximal stimulatory concentration for release is cumbersome and time consuming. Initial characterization of the underlying pharmacology, i.e. inhibitor versus substrate, followed by dose-dependent release assays performed in rat brain synaptosomes as described in various publications by Baumann and co-workers (Baumann et al., 2012; Baumann et al., 2013), has proven to be a reliable and time-efficient strategy. Moreover, assays conducted in native tissues serve as an important physiologically relevant comparator system, since the preparations closely reflect the natural MAT environment; apart from synaptosomes, this also includes slice preparations from brain tissue (Scholze, Norregaard, Singer, Freissmuth,

Gether & Sitte, 2002; Steinkellner et al., 2012).

21

Figure 5: Effect of AMPH in the absence and presence of mazindol on NET mediated reverse transport in HEK293 cells in the static-batch release assay. HEK293 cells stably expressing human NET were exposed to 50 nM of [3H]-MPP+ for 20 minutes. After washing the cells with buffer, the cells were exposed to buffer containing AMPH (2 μM) in presence or absence of mazindol (MAZ, 10 μM) for 10 minutes. Subsequently, the cells were lysed in 1 % SDS. For analysis, radioactivity present in the supernatant and the cell-lysate was determined by liquid scintillation counting. “Release” is expressed as percentage of total radioactivity present within one well, i.e. the sum of radioactivity present in the supernatant and the cell-lysate.

Data are shown as modified version of (Rosenauer, Luf, Holy, Freissmuth, Schmid & Sitte,

2013).

4 Biological assays to identify street drugs of unknown content To identify the mechanism of action for purchased stimulants that were classified as

“unknown” by ‘checkit!’, pharmacological “fingerprints” for well-established reference compounds were first generated. These reference fingerprints include concentration- 22 response curves for AMPH, MDMA, D-, methamphetamine, mephedrone,

MDPV and cocaine, obtained from uptake inhibition experiments performed with HEK293 cells expressing the human isoforms of MATs. Each substance reveals a unique profile of selectivity for uptake inhibition at DAT, NET and SERT (Figure 6). For example, the data in

Figure 6 demonstrate that methamphetamine displays potent inhibition of uptake at DAT and NET but not SERT, whereas methylone is much less selective in this regard. For the initial evaluation of an unknown compound, the test drug is examined for its ability to inhibit uptake in a dilution series covering six orders of magnitude. In addition to human DAT, NET and SERT, rat GABA transporter 1 (rGAT1) is included in the experimental series. rGAT1 serves as negative control since amphetamines do not function as substrates at this member of the neurotransmitter:sodium symporter family (Seidel et al., 2005). Data on uptake inhibition reveal the selectivity of test drugs for the individual MATs. Comparing the profile of activity for an unknown drug with the various reference “fingerprint” drugs allows for identifying the drug under investigation, or at least narrowing down the choice of potential candidate drugs. If amphetamine-like transporter substrates are suspected, static-batch release assays are performed to verify substrate-like activity at MATs. Similar to the uptake inhibition experiments, each drug is characterized for its ability to serve as a substrate at

DAT, NET and SERT. The combination of uptake inhibition and release experiments provides the basis to identify drugs based on their pharmacological fingerprints.

23

Figure 6: Uptake-inhibition profile of reference compounds in HEK293 cells expressing human monoamine transporters. HEK293 cells stably expressing the human isoforms of DAT,

NET and SERT were incubated with increasing concentrations of methamphetamine (A) or methylone (B) and tritiated substrate (SERT: 0.03 μM [3H]-5-HT; DAT and NET: 0.05 μM [3H]-

MPP+). The synopsis of the dose-response curves at each MAT reveals a unique profile of selectivity for each substance. Data are shown as modified versions of (Rosenauer, Luf, Holy,

Freissmuth, Schmid & Sitte, 2013).

5 Choosing the appropriate expression system Heterologous expression systems provide a powerful strategy to investigate MATs in vitro. These systems bypass the possible impact of vesicular storage mechanisms and presynaptic autoreceptors on the effects of test drugs. Furthermore, native tissue preparations normally contain more than one MAT. Hence, the use of specific inhibitors is a prerequisite to eliminate “off target” effects of test drugs when using tissue preparations.

24

Consequently, expression of MATs in heterologous systems ensures that the chosen MAT mediates the observed effects per se. However, it is noteworthy that a reduction in Na+/K+-

ATPase levels in non-neuronal cells and different membrane compositions can bias transporter function. Another important issue is choosing between stable or transient expression. Stable expression systems display constant expression levels. However, expression levels correlate with the relative potencies of test drugs (Ukairo, Ramanujapuram

& Surratt, 2007) and high expression levels have been shown to result in steep dose- response curves (Shoichet, 2006). To exclude data misinterpretation, it is critical to assess the kinetic parameters (KM and Vmax) of the chosen cell lines. Additionally, the inclusion of internal standard drugs with known pharmacology (e.g. cocaine) is a necessary step when estimating the relative potencies of new drugs.

6 Discussion Various methodologies have been employed to assess the mechanism of action of drugs at MATs. These include electrophysiological recordings (Sandtner et al., 2016); efflux of preloaded [3H]-substrates in static batch release assays (Wall, Gu & Rudnick, 1995) and native tissue preparations (Baumann et al., 2012; Crespi, Mennini & Gobbi, 1997); and dynamic superfusion experiments (Sitte, Scholze, Schloss, Pifl & Singer, 2000). The ultimate goal of applying these various methods is to identify the precise molecular mechanism of action for drugs interacting at MATS: uptake inhibitors versus substrates. Drug induced carrier-mediated release events have been observed and studied for over five decades

(Glowinski & Axelrod, 1965). However, the exact mechanism and subsequent intracellular cascades involved in transporter-mediated release are far from being fully understood.

Amphetamine-like transporter substrates can cause internalization events (Wheeler et al., 25

2015), alter the activity of kinases (Giambalvo, 1992) and even affect second messengers

(Gnegy et al., 2004) and the transcriptome (Allen, Wilkinson, Soo, Hui, Chase & Carrey,

2010). Hence, it is of tremendous importance to unravel the mode of action of NPS at plasmalemmal transporters as they serve as a gateway to the intracellular compartment.

Once located in the cytosol, NPS potentially trigger a variety of additional effects. For instance, a major target of amphetamine-like drugs appears to be VMAT2. Neurotoxicity of drugs can be linked to their substrate like-activity at the plasmalemmal and vesicular MATs

(Pifl, Reither & Hornykiewicz, 2015). After deciphering the mode of drug action at DAT, NET and SERT, substrates can be tested for their activities at VMAT2. The current problem with

NPS is that the drug markets are flooded with substances of unknown pharmacology. The rapid emergence and disappearance of NPS complicates long-term studies about their in vivo. Studies that examine the mode of action of psychostimulant NPS in vitro provide the basis to estimate the potential threat of individual drugs to public health. The techniques outlined in this chapter enable fast and reliable identification of MAT-substrates that require further analysis in more depth. Most importantly, the combination of biological assays and chemical identification by HPLC-MS by ‘checkit!’ is of tremendous clinical relevance. The current volatility of the street drug markets makes it cumbersome and challenging for medical professionals to treat adverse effects when the underlying pharmacology of ingested drugs is unknown. Maintaining an up-to-date database on the pharmacology and toxicology of newly-emerging drugs is essential for formulating effective responses to the problem of NPS. As outlined above, street drugs are rarely sold in their pure form. The current ‘checkit!’ warning system collects information on adulterants and potentially harmful drug-combinations. In addition, elucidating the chemical nature and pharmacological fingerprints for new drugs provides the fundamental knowledge required to

26 regulate and ban problematic drugs of abuse before they become well-established members of the drug market.

27

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Results  Prologue  Various clinically or recreationally used drugs give rise to bioactive metabolites. For instance, metabolism of MDMA (Green et al., 2014) or (Rothman et al., 2002) results in phase I metabolites that target MATs. However, in case of mephedrone, it was unknown if the phase I metabolites detected in plasma and urine (Meyer et al., 2010, Pedersen et al., 2013) mimic the action of the parental compound mephedrone, which acts as amphetamine-like releaser of monoamines (Baumann et al., 2012).

To assess the potential activity of the phase I metabolites in vitro, I performed uptake inhibition and release assays in HEK293 cells that expressed human DAT, NET or SERT, respectively. In presence of increasing concentrations of racemic mixtures of mephedrone and its metabolites, inwardly directed transporter assays revealed a clear concentration-dependent uptake inhibition for each of the tested metabolites. The metabolites 4-methylcathinone (nor-mephedrone) and 4-hydroxytolyl- mephedrone (4-OH-mephedrone) inhibited uptake mediated by DAT and NET such that they were roughly comparable to mephedrone with IC50 values in the range of 0.7 up to 7 µM. At SERT, nor-mephedrone exerted similar effects as mephedrone

(IC50 values of 10.6 and 7.8 µM, respectively), whereas 4-OH-mephedrone was weak

(IC50 > 70 µM). The reduced keto-metabolite (dihydro-mephedrone) showed only moderate effects as an uptake inhibitor at DAT, NET and SERT, with IC50 values greater than 20 µM. Outwardly directed transport assays in HEK293 cells showed that the metabolites acted as substrates of DAT, NET and SERT, as the presence of the metabolites induced efflux of via MATs. This interpretation is bolstered by the observation that monensin, which disrupts the sodium gradient and thus selectively enhances substrate-induced efflux (Scholze et al., 2000), augmented metabolite- induced efflux at each transporter.

The in vitro findings could be verified in ex vivo and in vivo experiments, performed in the group of Dr. Michael H. Baumann. Release assays performed in rat brain synaptosomes showed that nor- and 4-OH-mephedrone act as full-fledged releasers at MATs in a concentration-dependent manner. Most importantly, as assessed by in

RT vivo microdialysis, systemic administration of nor-mephedrone triggered the release of DA and 5-HT in the nucleus accumbens and induced locomotor activity in a dose- dependent manner. In the same experimental paradigm, systemic administration of 4-OH-mephedrone did not affect monoamine release or behavior to a significant extent. The data presented in this study show that the mephedrone-metabolites induce transporter-mediated release in vitro and that nor-mephedrone shows central activity upon systemic administration. However, the data do not allow estimating the potential contribution of the metabolites to the effects of mephedrone in vivo, since detailed information on pharmacokinetics of the metabolites is not available at present.

Dr. Laurin Wimmer synthesized the metabolites in the group of Prof. Dr. Marko D. Mihovilovic. A detailed description of the synthesis is available online in the supporting information: http://dx.doi.org/10.1111/bph.13547

Publication

Felix P. Mayer, Laurin Wimmer, Ora Dillon-Carter, John S. Partilla, Nadine V. Burchardt, Marko D. Mihovilovic, Michael H. Baumann*, Harald. H. Sitte* (2016); “Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters”; British Journal of Pharmacology, 173(17):2657-68. doi: 10.1111/bph.13547.

SK British Journal of British Journal of Pharmacology (2016) •• ••–•• 1 BJP Pharmacology RESEARCH PAPER Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters

Correspondence Harald S. Sitte, Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Währingerstrasse 13A, 1090 Vienna, Austria. E-mail: [email protected]

Received 24 February 2016; Revised 30 May 2016; Accepted 26 June 2016

FPMayer1, L Wimmer2, O Dillon-Carter3, J S Partilla3,NVBurchardt1, M D Mihovilovic2,MHBaumann3*and HHSitte1,4*

1Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Vienna, Austria, 2Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria, 3Designer Drug Research Unit (DDRU), Intramural Research Program (IRP), NIDA, NIH, Baltimore, MD, USA, and 4Center for Addiction Research and Science, Medical University Vienna, Vienna, Austria

*Equal contribution.

BACKGROUND AND PURPOSE 4-Methyl-N-methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate-type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5-HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N-demethylated metabolite, 4-methylcathinone (nor-mephedrone); the ring-hydroxylated metabolite, 4-hydroxytolylmephedrone (4-OH-mephedrone); and the reduced keto-metabolite, dihydromephedrone. EXPERIMENTAL APPROACH We used in vitro assays to compare the effects of mephedrone and synthetically prepared metabolites on transporter-mediated uptake and release in HEK293 cells expressing human monoamine transporters and in rat brain synaptosomes. In vivo microdi- À alysis was employed to examine the effects of i.v. metabolite injection (1 and 3 mg·kg 1) on extracellular dopamine and 5-HT levels in rat nucleus accumbens. KEY RESULTS In cells expressing transporters, mephedrone and its metabolites inhibited uptake, although dihydromephedrone was weak overall. In cells and synaptosomes, nor-mephedrone and 4-OH-mephedrone served as transportable substrates, inducing release via monoamine transporters. When administered to rats, mephedrone and nor-mephedrone produced elevations in extracellular dopamine and 5-HT, whereas 4-OH-mephedrone did not. Mephedrone and nor-mephedrone, but not 4-OH-mephedrone, induced locomotor activity. CONCLUSIONS AND IMPLICATIONS Our results demonstrate that phase I metabolites of mephedrone are transporter substrates (i.e. releasers) at DAT, NET and SERT, but dihydromephedrone is weak in this regard. When administered in vivo, nor-mephedrone increases extracellular dopamine and 5-HT in the brain whereas 4-OH-mephedrone does not, suggesting the latter metabolite does not penetrate the blood–brain barrier. Future studies should examine the pharmacokinetics of nor-mephedrone to determine its possible contribution to the in vivo effects produced by mephedrone.

Abbreviations 4-OH-mephedrone, 4-hydroxytolylmephedrone; DAT, dopamine transporter; GBR12935, 1-(2-diphenylmethoxyethyl)- 4-(3-phenylpropyl) dihydrochloride; MPP+, 1-methyl-4-phenylpyridinium; NET, noradrenaline transporter; nor-mephedrone, 4-methylcathinone; PDL, poly-D-lysine; SERT, 5-HT transporter

© 2016 The Authors. British Journal of Pharmacology DOI:10.1111/bph.13547 published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. F P Mayer et al. BJP

Tables of Links

TARGETS LIGANDS

a b Transporters Enzymes Amphetamine MDMA DAT, SLC6A3 CYP2D6 Citalopram MPP+ NET, SLC6A2 Cocaine SERT, SLC6A4 Desipramine Noradrenaline VMAT2, SLC18A2 Dopamine 5-HT GBR12935

These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS (Southan et al., 2016), and are permanently archived in the Concise Guide to a,b PHARMACOLOGY 2015/16 ( Alexander et al., 2015a,b).

Introduction neurotransmitter molecules from the extracellular space and move them back into the neuronal cytoplasm (i.e. up- During the past decade, a variety of man-made ‘designer take), thus terminating monoamine signalling (Kristensen drugs’ or ‘new psychoactive substances’ (NPS) have appeared et al., 2011; Reith et al., 2015). Drugs that interact with in the recreational drug market as legal alternatives to more DAT, NET and SERT can be classified as either cocaine-like traditional drugs of abuse (Baumann et al., 2014; Sitte and ‘blockers’ or amphetamine-like ‘substrates’ (Rothman and Freissmuth, 2015). Frequently, the chemical structures of Baumann, 2003; Sitte and Freissmuth, 2015). Both types NPS are based on known illicit substances and mimic their of compounds disrupt transporter function and produce el- psychoactive effects, but subtle structural modifications to evations in extracellular monoamine concentrations, but the drug molecules render them legal (Baumann and Volkow, their precise modes of action are different. On a molecular 2016). In particular, a number of NPS have been marketed as level, cocaine-like blockers act as non-transported inhibi- replacements for illicit stimulants like cocaine and 3,4- tors of monoamine transporters. Consequently, blockers methylenedioxymethamphetamine (MDMA, ‘ecstasy’) prevent the transporter-mediated uptake of released neuro- (Green et al., 2014). One of the most popular synthetic stim- transmitters from the extracellular medium. In addition, ulants is the cathinone analogue, 4-methyl-N- cocaine is known to mobilize the intracellular reserve pool methylcathinone or mephedrone. Mephedrone first ap- of dopamine and stimulate its exocytotic release (Venton peared in Israel as a ‘party drug’ during the early 2000s, and et al., 2006). In contrast, amphetamine-like compounds its recreational use spread to Europe, Australia and other parts are transported substrates that not only act as competitive of the world (Kelly, 2011). In the United States, mephedrone uptake inhibitors but also trigger neurotransmitter efflux was a constituent of so-called bath salts products, which be- by a complex process involving reversal of transporter flux came popular during 2010–2011 (Spiller et al., 2011). Low (Chen and Reith, 2004; Reith et al., 2015; Sitte and doses of mephedrone produce typical stimulant effects in Freissmuth,2015).Consequently,drugsthatactastrans- humans, like increased energy and mood elevation porter substrates are often referred to as ‘releasers’ as they (Vardakou et al., 2011; Winstock et al., 2011), while high induce a transporter-mediated efflux of neurotransmitters. doses or chronic use can produce life-threatening side ef- Studies using in vitro transporter assays in cells and rat fects including tachycardia, , agitation and brain synaptosomes have shown that mephedrone acts as a (James et al., 2011; Wood et al., 2011). Deaths from non-selective substrate at DAT, NET and SERT, thereby lead- mephedrone are rare but have been reported (Loi et al., ing to efflux of dopamine, noradrenaline and 5-HT 2015). In the interest of public health and safety, legisla- (Baumann et al., 2012; Eshleman et al., 2013; Simmler et al., tion was passed in many countries to ban the sale, posses- 2013). Systemic administration of mephedrone to rats in- sion and use of mephedrone (Drug Enforcement creases the extracellular concentrations of dopamine and 5- Administration, 2011; Green et al., 2014). Despite such HT in the brain, with the effects on 5-HT being somewhat bans, mephedrone continues to be abused in European greater in magnitude (Kehr et al., 2011; Baumann et al., countries (Archer et al., 2014; Hondebrink et al., 2015; 2012; Wright et al., 2012). Overall, the available preclinical EMCDDA, 2014). data indicate that mephedrone displays neurochemical ef- Similar to other stimulant drugs, mephedrone exerts its fects that mimic MDMA, but mephedrone has a number of effects by interacting with plasma membrane monoamine physiological and toxicological properties that render it transporter proteins of the solute carrier 6 family (SLC6) unique (Baumann et al., 2012; Miller et al., 2013; Shortall (Hadlock et al., 2011; Baumann et al., 2012; Martinez- et al., 2013). For example, high-dose administration of Clemente et al., 2012), namely the dopamine transporter mephedrone is less apt to produce robust hyperthermia and (DAT, SLC6A3), noradrenaline transporter (NET, SLC6A2) long-term depletions of brain tissue 5-HT (Baumann et al., and 5-HT transporter (SERT, SLC6A4). The normal role of 2012; den Hollander et al., 2013; Motbey et al., 2012), effects monoamine transporters is to capture previously released that are well established for MDMA. Importantly,

2 British Journal of Pharmacology (2016) •• ••–•• Bioactive metabolites of mephedrone BJP mephedrone has greatly reduced potency at the vesicular Methods monoamine transporter 2 (VMAT2, SLC18A2) when com- pared with MDMA and other ring-substituted amphetamines Animals and housing fl (Eshleman et al., 2013; Pi et al., 2015), suggesting Male Sprague Dawley rats from Harlan Laboratories (Freder- mephedrone is less likely to disrupt intracellular stores of ick, MD, USA) weighing 250–300 g at arrival were housed monoamine transmitters. three per cage for 2 weeks prior to being used in experiments. One possible explanation for the distinct effects of The rats were housed under standard conditions (lights on mephedrone is that metabolites of the drug contribute to from 0700–1900 h) with food and water available ad libitum. fi fi its in vivo pro le of actions. Meyer et al. (2010) rst Rats were maintained in facilities fully accredited by the Asso- reported that mephedrone is metabolized by three main ciation for Assessment and Accreditation of Laboratory Ani- hepatic mechanisms (Figure 1): (i) N-demethylation to mal Care (AAALAC), and experiments were performed in form 4-methylcathinone or nor-mephedrone; (ii) hydroxyl- accordance with the Institutional Care and Use Committee ation of the 4-methyl ring-substitution to form 4- of the NIDA Intramural Research Program. Rats used for brain hydroxytolylmephedrone (4-OH-mephedrone); and (iii) re- tissue harvest to prepare synaptosomes were housed in pairs, β duction of the -keto-oxygen group, which forms whereas those used in microdialysis experiments were dihydromephedrone (Meyer et al., 2010). Pedersen and co- housed singly post-operatively (see below). fi workers (2013) identi ed cytochrome P450 2D6 (CYP2D6) Animal studies are reported in compliance with the AR- as the main enzyme responsible for the phase 1 metabo- RIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, lism of mephedrone in humans and detected nor- 2015). A total of 16 rats was used for the in vitro synaptosome mephedrone, 4-OH-mephedrone and dihydromephedrone assays, and an additional 28 rats were used for in vivo microdi- in human urine specimens (Pedersen et al., 2013). As alysis experiments. pointed out by Green et al. (2014), no studies have exam- ined the pharmacology of mephedrone metabolites. There- fore, in the present investigation, we used in vitro assays to Cell culture compare the effects of mephedrone and its metabolites on The generation of HEK293 cells stably expressing the human transporter-mediated uptake and release in cells expressing isoforms of DAT (hDAT) and NET (hNET) was carried out as human DAT, NET and SERT and in rat brain synaptosomes. described previously (Scholze et al., 2002). For SERT, the hu- Additionally, the in vivo neurochemical effects of man isoform (hSERT) was cloned in frame with yellow fluo- systemically administered mephedrone, nor-mephedrone rescent protein (Schmid et al., 2001). The generation of a or 4-OH-mephedrone were examined using microdialysis stable cell line was performed as described by Hilber and col- in rat nucleus accumbens. Our data show that phase I me- leagues (Hilber et al., 2005). HEK293 cells were maintained in tabolites of mephedrone are substrates at monoamine humidified atmosphere (5% CO2, 37°C) in DMEM, transporters when assessed in vitro,butonlynor- supplemented with 10% heat-inactivated FBS and penicillin À À mephedrone displays substantial neurochemical actions (100 u 100 mL 1) and streptomycin (100 μg100mL1). in vivo, which could contribute to the behavioural effects Selection pressure was maintained by adding geneticin À of systemically administered mephedrone. (50 μg·mL 1) to the cell culture media.

Figure 1 Proposed pathways for the metabolism of mephedrone to its phase I metabolites. (i) demethylation forms 4-methylcathinone (NOR-MEPH); (ii) benzylic oxidation forms 4-hydroxytolylmephedrone (4-OH-MEPH); (iii) carbonyl reduction forms dihydromephedrone (DIHYDRO-MEPH). Chemical synthesis started from non-chiral precursors for the generation of racemic NOR-MEPH and 4-OH-MEPH and from chiral precursors for DIHYDRO-MEPH (racemic diastereomers obtained by mixing of ).

British Journal of Pharmacology (2016) •• ••–•• 3 F P Mayer et al. BJP

Transporter uptake assays in HEK293 cells [nomifensine and 1-(2-diphenylmethoxyethyl)-4-(3-phen- Uptake experiments were conducted as described previously ylpropyl)piperazine dihydrochloride (GBR12935) for SERT; (Sitte et al., 2001) with minor modifications. In brief, GBR12935 and citalopram for NET; citalopram and desipra- 3 + 3 HEK293 cells expressing hDAT, hNET or hSERT were seeded mine for DAT] to prevent the uptake of [ H]-MPP or [ H]-5- into poly-D-lysine (PDL) coated 96-well plates at a density of HT by competing transporters. Synaptosomes were preloaded 40 000 cells per well. The next day, DMEM was aspirated with radiolabelled substrate in Krebs-phosphate buffer, andreplacedwithKrebsHEPESbuffer(KHB,25mMHEPES, which consisted of 126 mM NaCl, 2.4 mM KCl, 0.5 mM

120mMNaCl,5mMKCl,1.2mMCaCl2,and1.2mM KH2PO4,1.1mMCaCl2,0.83mMMgCl2,0.5mMNa2SO4, À1 MgSO4, 5 mM D-glucose, pH adjusted to 7.3 with NaOH) 11.1 mM glucose, 13.7 mM Na2HPO4, 1 mg·mL ascorbic (200 μL per well), and cells were pre-incubated with various acid and 50 μM pargyline (pH = 7.4) for 1 h (steady state). As- concentrations of mephedrone or its metabolites for 5 min says were initiated by adding 850 μLofpreloaded (50 μL per well). Subsequently, 0.1 μMof[3H]-5-HT or synaptosomes to 150 μLoftestdrug.Dose–response curves 0.02 μMof[3H]-MPP+ were added, and uptake was terminated were generated using eight different concentrations of after 1 (hSERT) or 3 min (hDAT, hNET) by washing the cells mephedrone, nor-mephedrone or 4-OH-mephedrone. Assays with 200 μL of ice-cold KHB. Cells were lysed with 1% SDS, were terminated by vacuum filtration, and retained radioac- and tritium uptake was determined by scintillation counting. tivity was quantified by liquid scintillation counting. Nonspecific uptake was determined in the presence of 10 μM paroxetine (hSERT) or 10 μM mazindol (hDAT and hNET). Microdialysis in rat nucleus accumbens In vivo microdialysis procedures were carried out as previously fi Transporter release assays in HEK293 cells described with minor modi cations (Baumann et al., 2012). Briefly, male rats anaesthetized with sodium pentobarbital Superfusion experiments were performed as described previ- À (60 mg·kg 1, i.p.) received surgically implanted jugular ously (Scholze et al., 2002). Briefly, HEK293 cells expressing catheters, and intracerebral guide cannulae aimed at the nu- the desired transporter were seeded at a density of 40 000 cells cleusaccumbens(AP+1.6mm,MLÀ1.7 mm relative to per well onto poly-D-lysine-coated 5 mm glass cover slips in bregma; À6.2 mm relative to dura) (Paxinos and Watson, 96-well plates 24 h prior to the experiment. Cells were 2007). After a 7–10 day recovery, each rat was placed into a preloaded with [3H]-MPP+ (0.1 μM, hDAT and hNET) or chamber equipped with photobeams for the detection of mo- [3H]-5-HT (0.4 μM, hSERT) for 20 min at 37°C in a final vol- tor parameters (TruScan, Harvard Apparatus, Holliston, MA, ume of 100 μL per well. Subsequently, glass coverslips were USA) and allowed to acclimatize overnight. Food and water transferred into small superfusion chambers (volume of were available ad libitum during the acclimatization period. 200 μL) and superfused with KHB at 25°C with a superfusion À On the following morning, catheters were attached to PE 50 rate of 0.7 mL·min 1 for 40 min to establish a stable basal ef- extension tubes, and 0.5 × 2 mm microdialysis probes flux. After washout, the collection of 2-min fractions was ini- (CMA/12, Harvard Apparatus, Holliston, MA, USA) were tiated. After the first three basal fractions, monensin (10 μM) inserted into the guide cannulae. Ringers’ solution (150 mM or solvent was added for four fractions. Consequently, the NaCl, 2.8 mM KCl and 2.0 mM CaCl2) was perfused through cells were challenged with test drugs (10 μM) for five fractions À the probes at 0.6 μL·min 1 for 3 h. To commence in the presence or absence of monensin. Finally, the cells were experiments, dialysate samples (20 μL) were collected at lysed in 1% SDS to determine the total radioactivity. Radioac- 20 min intervals, and drug or saline treatments were given af- tivity per fraction was assessed by a liquid scintillation coun- ter three baseline samples were obtained. Rats received two ter and expressed as fractional release, that is, the percentage 3 3 sequential i.v. injections of mephedrone or its metabolites, of released Hinrelationtototal H present at the beginning À with 1 mg·kg 1 administered at time zero, followed by of the fraction (Sitte et al., 2000). For analysis, release was À 3mg·kg 1 60 min later. Saline was administered using the expressed as AUC. AUC was calculated for t = 6 to 26 min same schedule in a separate group of rats. Dialysate concen- and normalized to basal efflux,thatis,t=0to4min. trations of dopamine and 5-HT were quantified using HPLC coupled to electrochemical detection (Baumann et al., Transporter release assays in rat brain 2012). Chromatographic data were exported to an Empower synaptosomes software system (Waters, Inc., Milford, MA, USA) for peak The ability of mephedrone and its metabolites to evoke re- identification, integration and analysis. lease via DAT, NET and SERT was determined in rat brain syn- Correct probe placements were assessed after the microdi- aptosomesaspreviouslydescribed(Baumannet al., 2012). alysis experiments. Rats were killed by CO2 narcosis then de- Rats were killed with CO2,decapitated,andbrainswere capitated. Brains were quickly removed and immersion fixed rapidly removed and dissected on ice. Synaptosomes were in 10% paraformaldehyde for 1 week. Subsequently, brains prepared from striatum for DAT assays, whereas synapto- were sectioned on a cryostat, and the location of each probe somes were prepared from whole brain minus striatum and tip was verified by inspection of photographic images of the cerebellum for the NET and SERT assays. [3H]-MPP+ (9 nM) brain taken with a digital camera using the macro lens wasusedastheradiolabelledsubstrateforDATandNET, setting. whereas [3H]-5-HT (5 nM) was used as the radiolabelled substrate for SERT. All buffers used in the release assays Analysis contained 1 μM reserpine to block vesicular uptake of sub- Calculations were performed using Microsoft Excel® 2010 strates. The selectivity of assays was optimized for a single (Microsoft Corporation, Redmond, WA, USA) and GRAPHPAD transporter by including unlabelled compounds PRISM 5.0. (GraphPad Software Inc., La Jolla, CA, USA). IC50

4 British Journal of Pharmacology (2016) •• ••–•• Bioactive metabolites of mephedrone BJP

values for uptake inhibition and EC50 values for release were Synthetic procedures and chemical characterization data are determined by nonlinear regression fits. Release data given in detail in the Supporting Information. Reagents used expressed as AUC were analysed by one-way ANOVA in the experiments for uptake inhibition and release in followed by Bonferroni’s multiple comparison test. Microdi- HEK293 cells were used as mentioned in Hofmaier et al., alysis and locomotor data were analysed by two-way ANOVA 2014. Plasmids encoding human SERT were a generous gift (drug treatment × time) followed by Bonferroni’stest.Theef- of Dr Randy D. Blakely. For uptake and release experiments fect of monensin treatment on basal efflux of tritiated sub- in HEK293-cells and rat brain synaptosomes, [3H]-1-methyl- À strate was analysed with the Mann–Whitney test. P values 4-phenylpyridinium ([3H]-MPP+;80–85 μCi mmol 1)and À less than 0.05 (i.e. P < 0.05) were considered significant. [3H]-5-HT (28.3 μCi mmol 1) were purchased from American The data and statistical analysis comply with the recommen- Radiolabeled Chemicals (St. Louis, MO, USA) and Perkin dations on experimental design and analysis in pharmacol- Elmer (Boston, MA, USA) respectively. All other chemicals ogy (Curtis et al., 2015). and cell culture supplies were from Sigma-Aldrich (St. Louis, MO, USA) with the exception of cell culture dishes, which Materials were obtained from Sarstedt (Nuembrecht, Germany). 2-Methylamino-1-(p-tolyl)propan-1-one hydrochloride (me- phedrone, MW: 213.70), 2-amino-1-(p-tolyl)propan-1-one hydrochloride (nor-mephedrone, MW: 199.68) and 1-(4-(hy- Results droxymethyl)phenyl)-2-(methylamino)propan-1-one hydro- chloride (4-OH-mephedrone, MW: 229.70) were synthesized Mephedrone metabolites inhibit as racemic mixtures. In the case of 2-(methylamino)-1-(p- transporter-mediated uptake in HEK293 cells tolyl)propan-1-ol hydrochloride (dihydromephedrone, MW: We first tested the effects of mephedrone and its metabolites on 215.72), all four stereoisomers [syn-(1R,2R), syn-(1S,2S), transporter-mediated uptake. Figure 2 shows that mephedrone, anti-(1R,2S) and anti-(1S,2R)] were synthesized in their nor-mephedrone, 4-OH-mephedrone and dihydromephedrone enantiopure form (99%) and tested as 1:1:1:1 mixture. were fully efficacious inhibitors of uptake in HEK293 cells stably

Figure 2 Effects of mephedrone (MEPH), nor-mephedrone (NOR-MEPH), 4-OH-mephedrone (4-OH-MEPH) and dihydromephedrone (DIHYDRO) on trans- porter-mediated uptake in HEK293 cells expressing hDAT, hNET and hSERT. Uptake of [3H]-MPP+ via hDAT and hNET, and uptake of [3H]-5-HT by hSERT, was performed as described in Methods; all symbols represent mean values ± SEM, and the numbers in parentheses indicate the number of individual experiments performed in triplicate: hDAT: MEPH (3), NOR-MEPH (4), 4-OH-MEPH (4), DIHYDRO-MEPH (3); hNET: MEPH (4), NOR- MEPH (4), 4-OH-MEPH (3), DIHYDRO-MEPH (4); hSERT: MEPH (3), NOR-MEPH (3), 4-OH-MEPH (3), DIHYDRO-MEPH (3).

British Journal of Pharmacology (2016) •• ••–•• 5 F P Mayer et al. BJP expressing hDAT, hNET and hSERT. The potency of nor- mephedrone on [3H]-MPP+ efflux.Additionally,monensin mephedrone and 4-OH-mephedrone to inhibit [3H]-MPP+ up- alone elicited a significant albeit modest increase in substrate take via hDAT and hNET was comparable with mephedrone, release (P < 0.05, Mann–Whitney test), in agreement with our with IC50 values in the low micromolar range, from 0.7 to previous publications (Scholze et al., 2000). As a means to 6 μM. The IC50 values for dihydromephedrone to inhibit uptake summarize the overall effect of test drugs on release, with via hDAT and hNET were much weaker (i.e. 24 μM). Uptake and without monensin (10 μM), the data in Figure 3B–Dare inhibition experiments carried out with hSERT-expressing cells expressed as AUC for the nine fractions collected after drug revealed that nor-mephedrone inhibited uptake in the low treatment. One-way ANOVA demonstrated that monensin 3 + micromolar range with an IC50 value of 10.6 μM, whereas significantly influenced the release of [ H]-MPP evoked by 4-OH- and dihydromephedrone were much less active with mephedrone and its metabolites at DAT (F7,91 =24.61, IC50 values exceeding 60 μM. The obtained IC50 values are P < 0.001) and NET (F7,85 =14.4,P < 0.001). Post hoc analysis shown in Table 1. revealed that enhancement by monensin was significant for mephedrone, nor-mephedrone and 4-OH-mephedrone at Mephedrone metabolites induce hDAT and hNET, but not for dihydromephedrone. One-way transporter-mediated release in HEK293 cells ANOVA demonstrated that monensin significantly aug- 3 < Data from uptake inhibition assays cannot distinguish mented the release of [ H]-5-HT (F7,71 = 31.68, P 0.001) whether test drugs act as non-transported inhibitors or trans- via hSERT, and this effect was significant for mephedrone portable substrates, which evoke release (Scholze et al., 2000; and all of its metabolites. Sitte et al., 2000; Baumann et al., 2013). Therefore, mephedrone and its metabolites were tested in release assays Mephedrone metabolites induce to further explore their interaction with transporters. The re- transporter-mediated release in synaptosomes lease assays were performed with the same transporter- Next, we examined the effects of mephedrone and its me- expressing HEK293 cell lines described above and used a tabolites in rat brain synaptosomes to (i) analyse effects of superfusion system (Sitte et al., 2000). As described previ- test compounds in a native tissue preparation that contains ously, efflux of preloaded [3H]-MPP+ or [3H]-5-HT was moni- plasma membrane transporters in situ and (ii) compare data tored in the presence or absence of monensin (10 μM) from the human and rat transporters. Mephedrone, nor- (Scholze et al., 2000). Monensin acts as a selective H+/Na+ ion- and 4-OH-mephedrone were tested in release assays in rat ophore and dissipates the Na+ gradient across cell membranes brain synaptosomes, under conditions, which were opti- (Mollenhauer et al., 1990). This compound increases the in- mized for each transporter as described previously tracellular Na+-concentration (Chen and Reith, 2004) and (Baumann et al., 2012). The dose-effect release data are thus selectively enhances efflux triggered by transporter sub- depicted in Figure 4, and the calculated EC values are strates. Importantly, only substrate-induced release will be 50 shown in Table 2. In comparison with the parent com- enhanced by the application of monensin, while the effects pound mephedrone, nor- and 4-OH-mephedrone displayed of non-transported inhibitors will remain unchanged only slightly reduced potencies as releasers of preloaded (Scholze et al., 2000; Baumann et al., 2013; Sandtner et al., 3 + [ H]-MPP at DAT and NET, with EC srangingfrom 2016). The superfusion assays performed here are a decisive 50 0.05 μMto0.22μM (Figure 4 and Table 2). At SERT, nor- tool to discriminate between inhibitors and substrates 3 mephedrone induced release of preloaded [ H]-5-HT in a (Scholze et al., 2000). manner comparable with mephedrone (EC =0.2μM), Time-course experiments with mephedrone and its me- 50 whereas a 10-fold rightward shift was detected for 4-OH- tabolites (10 μM) demonstrated that all of the agents evoked mephedrone (EC =2μM). significant release of preloaded [3H]-MPP+ via hDAT and 50 hNET, and release of preloaded [3H]-5-HT via hSERT. Figure 3A depicts a representative example of time course ef- Nor-mephedrone, but not 4-OH-mephedrone, fects for DAT-mediated release of [3H]-MPP+ induced by nor- affects neurochemistry and behaviour in vivo mephedrone in the presence or absence of monensin. It is The findings from human and rat transporters agreed that clear that monensin markedly enhanced the effects of nor- mephedrone, nor-mephedrone and 4-OH-mephedrone were

Table 1

IC50 values of test drugs on uptake mediated by hDAT, hNET and hSERT, stably expressed in HEK293 cells

IC50 (μM) DAT NET SERT

Mephedrone 0.77 (0.53–1.08) 2.77 (1.92–3.97) 7.83 (6.32–9.75) Nor-mephedrone 6.35 (4.66–8.64) 5.46 (3.58–8.31) 10.61 (9.06–12.43) 4-OH-mephedrone 2.92 (2.35–3.6) 4.85 (3.28–7.17) 73.53 (62.5–86.51) Dihydromephedrone 23.97 (8.65–66.46) 23.53 (19.8–27.97) 64.98 (50.66–83.37) Data are represented as the mean with 95% confidence intervals in parentheses obtained from nonlinear regression fits as shown in Figure 2.

6 British Journal of Pharmacology (2016) •• ••–•• Bioactive metabolites of mephedrone BJP

Figure 3 Effects of mephedrone (MEPH), nor-mephedrone (NOR-MEPH), 4-OH-mephedrone (4-OH-MEPH) and dihydromephedrone (DIHYDRO-MEPH) on transporter-mediated release of preloaded radiolabelled substrate in HEK293 cells expressing hNET, hDAT and hSERT. [3H]-MPP+ was used as the radiolabelled substrate for hDAT and hNET while release by hSERT-expressing cells was performed using [3H]-5-HT as the radiolabelled sub- strate. (A) Representative experiment showing the effect of nor-mephedrone (10 μM) in the presence or absence of monensin (10 μM) on DAT- mediated efflux of pre-loaded [3H]-MPP+ (presence of substances indicated by black bar; n = 5 independent experiments performed in triplicate). (B–D) For each transporter, AUC was calculated from nine fractions collected after drug treatment (10 μM) in the absence or presence of monensin (MON, 10 μM). Solid bars indicate vehicle + drug, whereas hatched bars indicate MON + drug. Bars represent mean values ± SEM, and the numbers in parentheses indicate the number of individual experiments performed in triplicate: hDAT: MEPH (6), NOR-MEPH (5), 4- OH-MEPH (5), DIHYDRO-MEPH (5); hNET: MEPH (5), NOR-MEPH (5), 4-OH-MEPH (6), DIHYDRO-MEPH (5); hSERT: MEPH (5), NOR-MEPH (5), 4-OH-MEPH (5), DIHYDRO-MEPH (5). *P < 0.05 (Bonferroni’s) compared with corresponding vehicle + drug group. potent substrates at monoamine transporters. Thus, we locomotor stimulant, but both compounds significantly À sought to examine the neurochemical effects of these stimulated motor activity at the 3 mg·kg 1 dose. three compounds in vivo.Specifically, extracellular concentrations of dopamine and 5-HT were assessed by mi- crodialysis in the nucleus accumbens of freely-moving rats. Discussion As depicted in Figure 5, application of two-way ANOVA (drug treatment × time) demonstrated that drug treatments signifi- The aim of the present study was to determine the pharmaco- cantly influenced dialysate concentrations of dopamine logical effects of phase I metabolites of mephedrone and deci-

(F3,24 = 63.22, P < 0.001) and 5-HT (F3,24 = 83.83, pher their precise mode of action at monoamine transporters. P < 0.001). Post hoc tests revealed that mephedrone increased The synthetic cathinone mephedrone has been shown to act À dopamine after 1 mg·kg 1, whereas mephedrone and nor- as a non-selective, amphetamine-like substrate at mono- À mephedrone both elevated dopamine after 3 mg·kg 1. amine transporters, thereby triggering release of dopamine, 4-OH-mephedrone had no significant impact on dopamine noradrenaline and 5-HT into the extracellular space at either dose tested. Mephedrone and nor-mephedrone ele- (Baumann et al., 2012; Eshleman et al., 2013; Simmler et al., vated dialysate concentrations of 5-HT in a nearly identical 2013). The neurochemical effects of mephedrone mimic manner, with increases of 15-fold and 25-fold above baseline those of MDMA (Kehr et al., 2011; Baumann et al., 2012; À for the 1 and 3 mg·kg 1 doses respectively. Finally, drug treat- Wright et al., 2012), but mephedrone has a number of distinct ments significantly affected motor behaviour (F3,24 = 36.05, pharmacological effects when compared with MDMA and P < 0.001) such that mephedrone and nor-mephedrone other ring-substituted amphetamines (reviewed by Green increased activity whereas 4-OH-mephedrone did not. et al., 2014). Many therapeutic and abused stimulant drugs – Mephedrone was more potent than nor-mephedrone as a including diethylpropion, phendimetrazine and MDMA –

British Journal of Pharmacology (2016) •• ••–•• 7 F P Mayer et al. BJP

Figure 4 Effects of mephedrone (MEPH), nor-mephedrone (NOR-MEPH) and 4-OH-mephedrone (4-OH-MEPH) on transporter-mediated release of preloaded radiolabelled substrate in rat brain synaptosomes. [3H]-MPP+ was the radiolabelled substrate for DAT and NET assays while [3H]-5- HT was the radiolabelled substrate for SERT assays. Symbols represent mean values ± SEM obtained from three individual experiments performed in triplicate.

Table 2

EC50 values of test drugs on transporter mediated efflux obtained in rat brain synaptosomes

EC50 (μM) DAT NET SERT

Mephedrone 0.052 (0.036–0.075) 0.09 (0.08–0.11) 0.21 (0.17–0.26) Nor-mephedrone 0.22 (0.14–0.32) 0.1 (0.08–0.13) 0.21 (0.13–0.32) 4-OH-mephedrone 0.19 (0.13–0.267) 0.15 (0.11–0.19) 2.01 (1.390–2.91) Data are represented as the mean and 95% confidence intervals in brackets obtained from nonlinear regression fitsasshowninFigure4. are transformed by hepatic mechanisms into bioactive me- dependent manner at all three human plasma membrane mono- tabolites (Yu et al., 2000; Rothman et al., 2002; Green et al., amine transporters. Nor-mephedrone and 4-OH-mephedrone 2003). To examine whether metabolites of mephedrone inhibited uptake at hDAT and hNET with potency comparable might be bioactive, we tested the known metabolites nor- with mephedrone, whereas dihydromephedrone was much mephedrone, 4-OH-mephedrone and dihydromephedrone weaker. Uptake inhibition assays can identify compounds that for their interactions with DAT, NET and SERT. It was found interact with monoamine transporters, but cannot discrimi- that all of the metabolites acted as substrate-type releasers, nate whether such compounds act as inhibitors or substrates. but nor-mephedrone and 4-OH-mephedrone were much Thus, we tested the effects of mephedrone metabolites more potent than dihydromephedrone in this regard. Impor- using release assays in HEK293 cells and rat brain synapto- tantly, only nor-mephedrone influenced brain neurochemis- somes. Nor-mephedrone and 4-OH-mephedrone evoked re- try and behaviour upon systemic administration. lease of radiolabelled substrates from HEK293 cells stably The present in vitro data from HEK293 cells show that expressing hDAT, hNET or hSERT. The releasing action of mephedrone metabolites inhibit uptake in a concentration- the drugs was augmented in the presence of monensin,

8 British Journal of Pharmacology (2016) •• ••–•• Bioactive metabolites of mephedrone BJP

Figure 5 Effects of i.v. administration of mephedrone (MEPH), nor-mephedrone (NOR-MEPH) and 4-OH-mephedrone (4-OH-MEPH) or saline (SAL) on À neurochemistry and behaviour in rats undergoing microdialysis in the nucleus accumbens. Drugs were administered i.v. at 1 mg·kg 1 at time À zero, followed by 3 mg·kg 1 60 min later. Dopamine and 5-HT were detected by HPLC-EC as described in Methods. Forward locomotion (activity) was determined by photo-beam breaks. Data are presented as mean ± SEM, n = 6 rats in the control group (SAL) and n = 7 rats for all other groups (MEPH, NOR-MEPH and 4-OH-MEPH), arrows indicate time of drug administration. Individual symbols represent significant differences from sa- line-treated control at corresponding time points (P < 0.05; Bonferroni’s): * denotes significance of MEPH compared to saline, and # denotes sig- nificance of NOR-MEPH compared to saline. an ionophore that dissipates Na+ gradients across plasma the surface area for drug-protein interactions. Additionally, membranes. The enhancement of release by monensin pro- HEK293 cells are non-neuronal in origin and do not pos- vides crucial mechanistic evidence that mephedrone and its sess all critical components of the plasma membrane pro- metabolites function mainly as transporter substrates, not tein machinery that are present in neurons in vivo. merely as inhibitors and thus are capable of inducing re- Despite the different assay systems and methods employed lease of monoamines via their cognate transporters. Consis- here, all of the findings agree that mephedrone and its me- tent with the data in HEK293 cells, nor-mephedrone and 4- tabolites are substrates at monoamine transporters. OH-mephedrone induced release of [3H]-MPP+ via DAT and Even though the mephedrone metabolites tested acted as NET, and release of [3H]-5-HT via SERT, in rat brain synap- transporter substrates in vitro, only nor-mephedrone signifi- tosomes. Our findings with nor-mephedrone in synapto- cantly affected neurochemistry and behaviour in vivo.The somes agree with the recent findings of Hutsell et al. neurochemical profile of nor-mephedrone closely resembled (2016) who reported that stereoisomers of nor-mephedrone that of mephedrone at the doses tested in our study, but (i.e. stereoisomers of 4-methylcathinone) are non-selective nor-mephedrone had weaker effects on extracellular dopa- transporter substrates that evoke neurotransmitter release mine and locomotion. Thus, it seems nor-mephedrone dis- from synaptosomes in vitro. plays a more profile of activity than the parent Previous investigations have revealed that the corre- compound mephedrone. The reduced locomotor response sponding IC50 and EC50 values for a given drug to inhibit to nor-mephedrone as compared with mephedrone is proba- uptake or induce release may differ several-fold (Scholze bly linked to blunted dopaminergic effects of the metabolite, et al., 2000; Sitte et al., 2001). The apparent differences in because previous studies have shown that extracellular dopa- potency that we observed here for inhibition of uptake mine levels in the nucleus accumbens are tightly correlated

(IC50 values in the μM range) versus stimulation of release with the extent of motor activation produced by stimulant (EC50 values in the nM range) might be attributed to differ- drugs (Zolkowska et al., 2009; Baumann et al., 2011). Surpris- ent assay systems and methods used in our studies. For ex- ingly, the systemic administration of 4-OH-mephedrone ample, uptake assays in HEK293 cells use static incubation had no significant effect on extracellular neurotransmitters conditions, while release assays in HEK293 cells use in the brain or on behaviour. Taken together with the dynamic perfusion conditions. Comparing results from re- in vitro findings, our in vivo data with 4-OH-mephedrone sug- lease assays with HEK293 cells versus rat brain synapto- gest this metabolite may not penetrate through the somesisevenmoreproblematicbecausethelatter blood–brain barrier. The likelihood of substances to enter preparation consists of homogenized tissue that maximizes the brain is correlated with their size and lipid solubility

British Journal of Pharmacology (2016) •• ••–•• 9 F P Mayer et al. BJP

(van Bree et al., 1988; Waterhouse, 2003). Distribution coeffi- Acknowledgements cients calculated for mephedrone and its metabolites indicate a clear-cut separation of lipohilic mephedrone and nor- We thank Dr Oliver Kudlacek and Marion Holy for providing mephedrone on the one hand (logD7.4 = 1.39 and 1.29, re- HEK293-cells stably expressing the human serotonin trans- spectively) and hydrophilic 4-OH-mephedrone on the other porter and Dr Lars Richter for calculating the distribution co- hand (logD7.4 = 0.14). As a consequence, the increased hy- efficients of mephedrone and its metabolites. The research drophilicity of 4-OH-mephedrone, as compared with was supported by Austrian Research Fund/FWF grants F3506 mephedrone and nor-mephedrone, likely precludes the hy- and W1232 (H.H.S.), the Intramural Research Program of droxylated metabolite from entering the brain. We have the NIDA, NIH, grant DA000523-07 (M.H.B.), the FWF-DK noted a similar situation to the hydroxylated metabolites of MolTag, FWF W1232 (M.D.M.), and F.P.M. is a recipient of a MDMA, which are devoid of central activity when adminis- DOC-fellowship of the Austrian Academy of Sciences. tered systemically to rats (Schindler et al., 2014). Neverthe- less, there is increasing evidence that points to the presence of various CYPs in brain tissue. Even though the expression levels of CYPs in the brain are low when compared with Author contributions those in liver (Miksys and Tyndale, 2002), it is interesting F.P.M., O.D-C., J.S.P., L.W. and N.B. performed all experimental to speculate that in situ metabolism of mephedrone and work. F.P.M., L.W., M.D.M., M.H.B. and H.H.S. designed the ex- formation of phase 1 metabolites in brain could impact periments. F.P.M., M.H.B. and H.H.S. wrote the manuscript and on mephedrone action in vivo.Forinstance,CYP2D6has received significant input from all other authors. been detected in various regions of , including substantia nigra and hippocampus (Siegle et al., 2001). As a consequence, mephedrone metabolites could be formed in fl close proximity to monoamine transporters and thereby Con ict of interest contribute to the effects of mephedrone. At present, there H.H.S. has received honoraria for lectures and consulting is no evidence that formation of metabolites in the central from AbbVie, Lundbeck, MSD, Pfizer, Ratiopharm, Roche, nervous system is of any pharmacological relevance. Inter- Sanofi-Aventis and Serumwerk Bernburg (past 5 years). All estingly, the dihydroxy metabolite of MDMA, 3,4- other authors declare no conflicts of interest. dihydroxymethamphetamine, displays potent stimulatory effects on heart rate and blood pressure upon systemic ad- ministration (Schindler et al., 2014). Our results suggest that future investigations should examine the possible car- Declaration of transparency and diovascular effects of 4-OH-MEPH. The most abundant scientificrigour mephedrone metabolite detected in blood from forensic traffic cases was 4-OH-MEPH (Pedersen et al., 2013). In This Declaration acknowledges that this paper adheres to the fi two cases, the blood concentrations of parent drug:metabo- principles for transparent reporting and scienti crigourofpre- litewere28:2and29:9μg/kg. In a number of cases, trace clinical research recommended by funding agencies, publishers amounts of NOR-MEPH and DIHYDRO-MEPH were also de- and other organisations engaged with supporting research. tected in blood and urine samples, but 4-OH-MEPH was highest in urine, highlighting its hydrophilicity. The present data alone cannot clarify whether nor- mephedrone contributes to the psychoactive properties of References systemically administered mephedrone in animals or humans. Further studies are needed to determine the blood Alexander SP, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE et al. and brain concentrations of nor-mephedrone after (2015b). The Concise Guide to PHARMACOLOGY 2015/16: enzymes. mephedrone exposure. It is noteworthy that nor- Br J Pharmacol 172: 6024–6109. mephedrone is the most abundant metabolite of Alexander SP, Kelly E, Marrion N, Peters JA, Benson HE, Faccenda E mephedrone identified in rats (Khreit et al., 2013; Martinez- et al. (2015a). The Concise Guide to PHARMACOLOGY 2015/16: Clemente et al., 2013) whereas 4-OH-mephedrone is the ma- transporters. Br J Pharmacol 172: 6110–6202. jor metabolite in humans (Pedersen et al., 2013; Pozo et al., 2015). Currently, no information is available on the pharma- Archer JR, Dargan PI, Lee HM, Hudson S, Wood DM (2014). Trend cokinetics and of the metabolites after analysis of anonymised pooled urine from portable street urinals in fi mephedrone administration in either species. The collective central London identi es variation in the use of novel psychoactive substances. Clin Toxicol (Phila) 52: 160–165. results presented here demonstrate that phase I metabolites of mephedrone are non-selective transporter substrates at Baumann MH, Ayestas MA Jr, Partilla JS, Sink JR, Shulgin AT, Daley PF DAT, NET and SERT, similar to the parent compound. et al. (2012).Thedesignermethcathinoneanalogs,mephedroneand However, only nor-mephedrone affects neurochemistry methylone, are substrates for monoamine transporters in brain and behaviour when administered peripherally, suggesting tissue. Neuropsychopharmacology 37: 1192–1203. fi this metabolite could contribute signi cantly to the unique Baumann MH, Clark RD, Woolverton WL, Wee S, Blough BE, fi pro le of psychoactive effects produced by mephedrone. Rothman RB (2011). In vivo effects of amphetamine analogs reveal Further studies are warranted to examine this intriguing evidence for serotonergic inhibition of mesolimbic dopamine hypothesis. transmission in the rat. J Pharmacol Exp Ther 337: 218–225.

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Sitte HH, Freissmuth M (2015). Amphetamines, new psychoactive drugsandthemonoaminetransportercycle.TrendsPharmacolSci Supporting Information 36: 41–50.

Sitte HH, Hiptmair B, Zwach J, Pifl C, Singer EA, Scholze P (2001). Additional Supporting Information may be found in the on- Quantitative analysis of inward and outward transport rates in cells line version of this article at the publisher’s web-site: stably expressing the cloned human serotonin transporter: inconsistencies with the hypothesis of facilitated exchange diffusion. http://dx.doi.org/10.1111/bph.13547 – Mol Pharmacol 59: 1129 1137. Figure S1 Unless noted otherwise, all reagents were pur- Sitte HH, Scholze P, Schloss P, Pifl C, Singer EA (2000). chased from commercial suppliers and used without further Characterization of carrier-mediated efflux in human embryonic purification.

12 British Journal of Pharmacology (2016) •• ••–•• Interlude  At present, NPS occur on the global drug market to provide legal alternatives for scheduled drugs of abuse (Baumann and Volkow, 2016). By subtle chemical modification of regulated compounds, NPS evade laws that regulate the parental compounds (Baumann et al., 2014b). Recently, a modified version of the anorectic drug phenmetrazine, i.e. 3-fluoro-phenmetrazine (3-FPM), occurred on the drug market and has been detected in patients who required hospital care (Backberg et al., 2016). Phenmetrazine targets DAT, NET and SERT in an amphetamine-like fashion, thus induces efflux of monoamines. However, phenmetrazine clearly favours DAT and NET over SERT (Rothman et al., 2002). At present, information on the pharmacology of 3-FPM is rare (Blough, 2011, Blough, 2013), so this study was designed to characterize the activity of 3-FPM and the positional isomers 2- and 4- FPM at MATs. 2- and 4-FPM were included as these compounds might appear on the drug market in the near future. As shown in the embedded publication, entitled “Pharmacological characterization of fluorinated phenmetrazine "research chemicals"“, which has been submitted to Neuropharmacology, each FPM acts as amphetamine-like releaser at MATs. Further, since each FPM appears to favor DAT and NET over SERT, these “research chemicals” might bear the potential to be addictive (Sulzer, 2011). 2-, 3- and 4-FPM were provided by Dr. Simon Brandt. I performed uptake inhibition and release assays in HEK293 cells, stably transfected with human DAT, NET or SERT, respectively. Nadine V. Burchardt, a diploma student, assisted me in this regard. Release assays in rat brain synaptosomes were performed in the laboratories of Dr. Bruce E. Blough and Dr. Michael H. Baumann. Together with Prof. Harald H. Sitte and Dr.Simon Brandt, I planned and designed experiments. Further, I wrote the first draft of the manuscript with help from Prof. Dr. Harald H. Sitte. We received significant input from all other co-authors, especially Dr. Michael H. Baumann and Dr. Simon D. Brandt.

TN Publication  Felix P. Mayer, Nadine V. Burchardt, Ann M. Decker, John S. Partilla, Gavin McLaughlin, Pierce V. Kavanagh, Michael H. Baumann, Bruce E. Blough, Simon D. Brandt, Harald H. Sitte; “Pharmacological characterization of fluorinated phenmetrazine “research chemicals””; In revision, Neuropharmacology (first submission Sept 2016)

TO Elsevier Editorial System(tm) for Neuropharmacology Manuscript Draft

Manuscript Number:

Title: Pharmacological characterization of fluorinated phenmetrazine "research chemicals"

Article Type: Research Paper

Keywords: New psychoactive substances, legal high, phenmetrazine, monoamine transporter, amphetamine

Corresponding Author: Prof. Harald H Sitte, MD

Corresponding Author's Institution: Medical University Vienna

First Author: Felix P Mayer, MSc

Order of Authors: Felix P Mayer, MSc; Nadine V Burchardt; Ann M Decker, PhD; John S Partilla, B.A.; Gavin McLaughlin, MSc; Pierce V Kavanagh, PhD; Michael H Baumann, PhD; Bruce E Blough, PhD; Simon D Brandt, PhD; Harald H Sitte, MD

Abstract: A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market, and Internet evidence suggests misuse. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine (IC50 values < 2.5 μM), but display less potent effects at SERT (IC50 values >39 μM). Experiments directed at identifying transporter- mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na+/H+ ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. The combined findings indicate that FPM isomers act as substrates for monoamine transporters. This study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at transporters implicated in abuse and addiction.

Cover letter

Bruno Frenguelli Editor-in-chief     

Vienna, Monday, September 12, 2016

Dear Dr. Frenguelli,

Enclosed is our manuscript “                  ”, which we are submitting for consideration as a research paper in      .

A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS, also referred to as “research chemicals” or “RCs”, are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market, and Internet evidence suggests misuse. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), both in heterologously expressing cell lines but also in ex vivo synaptosomes. The combined findings indicate that FPM isomers act as substrates for monoamine transporters. This study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasing agents with marked potency at catecholamine transporters implicated in abuse and addiction.

We confirm that our article is original research which has not been previously published and has not been submitted for publication elsewhere while under consideration.

Thank you for your attention to our work; we look forward to hearing from you.

Sincere regards,

Harald H. Sitte Professor for Psychopharmacology

Highlights

                                  *Title page

Pharmacological characterization of fluorinated phenmetrazine “research chemicals”

Felix P Mayera, Nadine V Burchardta, Ann M Deckerb, John S Partillac, Gavin McLaughlind,e, Pierce V Kavanaghd, Michael H Baumannc, Bruce E Bloughb, Simon D Brandtf, Harald H Sittea,g,*

a Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13A, 1090, Vienna, Austria

b Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, USA

c Designer Drug Research Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

d Department of Life and Physical Sciences, School of Science, Athlone Institute of Technology, Dublin Road, Westmeath, Ireland

e Department of Pharmacology and Therapeutics, School of Medicine, Trinity Centre for Health Sciences, St. James’s Hospital, Dublin 8, Ireland

f School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK

g Center for Addiction Research and Science - AddRess, Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria

* Corresponding author

Contact details [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Correspondence Harald H Sitte, MD, Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Währingerstrasse 13A, 1090 Vienna, Austria E-mail: [email protected]

  *Abstract

Pharmacological characterization of fluorinated phenmetrazine “research chemicals”

A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market, and Internet evidence suggests misuse. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine

(IC50 values < 2.5 µM), but display less potent effects at SERT (IC50 values >39 µM). Experiments directed at identifying transporter-mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na+/H+ ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. The combined findings indicate that FPM isomers act as substrates for monoamine transporters. This study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at catecholamine transporters implicated in abuse and addiction.  *Manuscript Click here to view linked References

Pharmacological characterization of fluorinated phenmetrazine “research chemicals”

Felix P Mayera, Nadine V Burchardta, Ann M Deckerb, John S Partillac, Gavin McLaughlind,e, Pierce V Kavanaghd, Michael H Baumannc, Bruce E Bloughb, Simon D Brandtf, Harald H Sittea,g,*

a Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13A, 1090, Vienna, Austria

b Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, USA

c Designer Drug Research Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

d Department of Life and Physical Sciences, School of Science, Athlone Institute of Technology, Dublin Road, Westmeath, Ireland

e Department of Pharmacology and Therapeutics, School of Medicine, Trinity Centre for Health Sciences, St. James’s Hospital, Dublin 8, Ireland

f School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK

g Center for Addiction Research and Science - AddRess, Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria

* Corresponding author

Contact details [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Correspondence Harald H Sitte, MD, Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Währingerstrasse 13A, 1090 Vienna, Austria E-mail: [email protected]

+  Abstract (250 w):

A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market, and Internet evidence suggests misuse. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine

(IC50 values < 2.5 µM), but display less potent effects at SERT (IC50 values >39 µM). Experiments directed at identifying transporter-mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na+/H+ ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. The combined findings indicate that FPM isomers act as substrates for monoamine transporters. This study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at catecholamine transporters implicated in abuse and addiction.

Keywords (max. 6) New psychoactive substances, legal high, phenmetrazine, monoamine transporter, amphetamine

Abbreviations1

 +! (/)&/)$#$ $ '&'&  ' &  %' ,3-& $$'&)  ) ' &  ' 4&+)  $).)$$ ' &  ' &"$ !  '  &    ,  1. Introduction

Novel drugs of abuse, more formally known as new psychoactive substances (NPS), are appearing at a rapid pace on recreational drug markets worldwide (Welter-Luedeke and Maurer, 2016). NPS are designed to imitate the actions of known drugs of abuse (e.g. amphetamines), while circumventing current legal restrictions on controlled substances due to their unique chemical structures (Baumann et al., 2014). NPS are marketed and sold over the Internet under various names like “research chemicals”, “bath salts” or ”legal highs”, and there is legitimate concern about their negative impacts on public health (Tettey and Crean, 2015). Based on the surveillance model first developed by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), the United Nations Office on Drugs and Crime (UNODC) launched the Early Warning Advisory (EWA) in 2013 to monitor the appearance of NPS on a global scale. Recently, the EWA identified 3- fluorophenmetrazine (3-FPM) as an NPS on Internet websites, and information from drug user forums suggests the drug might have psychostimulant properties in human users. Notably, the parent compound phenmetrazine was once prescribed as an anorectic medication but was withdrawn from the market because of its abuse potential (Griffiths et al., 1976, Banks et al., 2013). The process of developing and manufacturing NPS hinges on easy access to the biomedical literature where multiple information sources, such as patent applications and medicinal chemistry journals, provide a rich source of ideas for clandestine chemists (Brandt et al., 2014). The preparation of 3-FPM was first published in 2011 as part of a patent application for morpholine-based compounds that were being investigated for a range of potential therapeutic applications (BLOUGH, 2011, Blough, 2013) . Similar to other psychostimulant drugs, phenmetrazine targets the plasma membrane monoamine transporters (MATs) expressed on nerve cells (Rothman et al., 2002). MATs, or neurotransmitter:sodium symporters, belong to the solute carrier 6 family of proteins (SLC6) and mediate the uptake of monoamine neurotransmitters from the extracellular space (Kristensen et al., 2011). Specific transporters for serotonin (SERT, SLC6A4), dopamine (DAT, SLC6A3) and norepinephrine (NET, SLC6A2) exploit the sodium gradient across cell membranes to drive the movement of monoamines from the synaptic cleft into the neuronal cytoplasm, thus tightly regulating the strength and the duration of monoamine signaling (Torres et al., 2003). MATs represent major targets for clinically relevant drugs (e.g., antidepressants) but also for a plethora of scheduled substances, like cocaine and amphetamine (“speed”) (Kristensen et al., 2011). All psychostimulants that target MATs increase the extracellular concentrations of monoamines in the central and peripheral nervous systems. However, the precise mode of drug action may be subdivided into cocaine-like “blockers” and amphetamine-like “releasers” (Sitte and Freissmuth, 2010). The former act as non-transported inhibitors, whereas releasers are transportable substrates which reverse the normal direction of transporter flux to trigger transporter-mediated efflux

-  of neurotransmitters (Torres et al., 2003). Because substrate-type releasers are transported through the transporter channel along with sodium ions, they induce inwardly-directed sodium currents, and at sufficiently high intracellular concentrations, these drugs can redistribute neurotransmitters from vesicular storage pools into the cytosol (Sulzer et al., 2005, Sitte and Freissmuth, 2010). As a consequence, the intracellular concentrations of free monoamine neurotransmitters and sodium cations build up at the inner side of the plasma membrane to enable transporter-mediated reverse transport (Sitte and Freissmuth, 2015). Previous research shows that phenmetrazine acts as a substrate-type releaser at DAT and NET, with much weaker effects at SERT (Rothman et al., 2002). Chemical modification of amphetamine- type stimulant drugs can produce profound changes in their profile of pharmacological effects. For instance, addition of a fluorine to the 4-position on the phenyl ring of amphetamine increases relative potency toward SERT versus DAT (Marona-Lewicka et al., 1995, Nagai et al., 2007, Baumann et al., 2011, Rickli et al., 2015). Limited information is available on the pharmacology of 3-FPM (BLOUGH, 2011, Blough, 2013), so we sought to characterize the molecular mode of action of this drug in cells expressing human transporters and in native tissue preparations from rat brain. Based on the available structure-activity data for amphetamine-type compounds, we hypothesized that addition of a fluoro substitution to the phenyl ring of phenmetrazine would enhance the potency of analogs toward SERT compared to DAT. In vitro uptake inhibition and efflux assays were used to elucidate the mechanism of action of 3-FPM at MATs, and to evaluate whether the drug induces transporter- mediated release consistent with an amphetamine-type action. Two positional isomers, 2-FPM and 4- FPM, were included in our study for comparison, because isomers of NPS often appear in the marketplace and present challenges from a clinical and forensic perspective (Brandt et al., 2015, Brandt et al., 2014, Elliott et al., 2013, Dinger et al., 2016, Marusich et al., 2016, McLaughlin et al., 2016).

.  2. Materials and Methods

2.1 Reagents and Chemicals 2-(2-Fluorophenyl)-3-methylmorpholine (2-FPM), 2-(3-fluorophenyl)-3-methylmorpholine (3-FPM) and 2-(4-fluorophenyl)-3-methylmorpholine (4-FPM) were prepared as fumarate salts and analytically characterized previously (McLaughlin et al., 2016) [3H]-5HT (28.3 µCi mmol-1) was purchased from PerkinElmer (Boston, MD, USA), and [3H]-MPP+ (80-85 µCi mmol-1) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA), respectively. All other chemicals and reagents, including cell culture supplies, were purchased from Sigma Aldrich. Cell culture dishes were from Sarstedt AG&Co., Nuembrecht, Germany.

2.2 Cell Culture Human embryonic kidney (HEK293) cells were maintained in Dulbecco’s Modified Essential Medium (DMEM), supplemented with 10% fetal calf serum, streptomycin (100 µg 100 mL-1) and penicillin -1 (100 I.U. 100 mL ) and kept in humidified atmosphere (5 % CO2, 37 °C). The generation of HEK293 cells stably expressing human (h) DAT and hNET is described elsewhere (Scholze et al., 2002). Human SERT was cloned in frame with monomeric GFP (mGFP) into a tetracycline inducible expression vector pcDNA 4/TO (Invitrogen by life-Technologies, Carlsbad, CA, USA). The generation of a stable cell lines was performed as described previously (Hilber et al., 2005). The selection of HEK293 cells stably expressing i) hDAT and hNET or ii) hSERT was executed by constantly adding geneticin (50 µg mL-1) or blasticidin (10 µg mL-1) and zeocin (300 µg mL-1), respectively. Expression of hSERT was induced with tetracycline (1 µg mL-1) 24 h prior to the experiment.

2.3 Radiotracer Uptake and Efflux Experiments in HEK293 Cells Uptake inhibition and efflux experiments were performed as described previously (Scholze et al., 2000). Briefly, for uptake experiments HEK293 cells expressing the desired transporter were seeded at a density of 4*10^4 cells per well on a poly-D-lysine (PDL) coated 96-well plate the day before the experiment. The cells were incubated for five min with various concentrations of 2-,3- or 4-FPM in

Krebs-HEPES-buffer (KHB, 25 mM Hepes, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM

MgSO4 and 5 mM D-glucose, pH= 7.3) to allow for equilibration before the addition of radiolabeled substrate. The radiolabeled substrate for DAT and NET was [3H]-MPP+ (20 nM) whereas the radiolabeled substrate for SERT was [3H]-5HT (100 nM). After incubation for 180 s (DAT and NET) or 60 s (SERT), radioactive substrate was aspirated and the cells were washed with ice-cold KHB to terminate uptake. Subsequently, the cells were lysed in sodium dodecylsulfate (SDS, 1 %; 200 µL per well) and the amount of radioactivity within each well was assessed by use of a liquid scintillation counter. Nonspecific uptake was determined in presence of 10 µM mazindol (DAT), desipramine

/  (NET) or paroxetine (SERT). For analysis, nonspecific uptake was subtracted from all values and uptake was expressed as percent of control uptake, i.e. uptake in absence of inhibitor, and plotted against increasing concentrations of inhibitor. For efflux experiments, 4*10^4 HEK293 cells expressing hDAT, hNET or hSERT were seeded onto PDL-coated 5mm glass-coverslips. The cells were pre-loaded with 0.1 µM [3H]-MPP+ (hDAT and hNET) or 0.4 µM [3H]-5HT (hSERT) in KHB for 20 min at 37 °C. Subsequently, the cells were transferred into small chambers with a volume of 200 µL and superfused with KHB (25 °C, 0.7 mL per minute) for 40 min to establish a stable baseline. At t=0, the experiment was started by collecting three two-minute fractions of superfusate to record the basal efflux, followed by the addition of monensin (10 µM) or solvent for four fractions. Finally, the cells were challenged with 2-, 3- or 4- FPM (5 µM) for five fractions and then lysed in 1% SDS. Radioactivity was determined by liquid scintillation counting and radioactivity per fraction was expressed as fractional release, i.e. the percentage of released [3H] versus the amount of [3H] present at the beginning of that fraction (Sitte et al., 2000).

2.4 Radiotracer flux experiments in rat brain synaptosomes All experiments utilizing animal tissue were performed in agreement with the ARRIVE guidelines. Male Sprague-Dawley rats (Charles River, Wilmington, MA, US) weighing 300-400 g were maintained in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and experiments were performed in accordance with the Institutional Animal Care and Use Committee at Mispro Biotech and the National Institute on Drug Abuse (NIDA),

Intramural Research Program (IRP). Rats were euthanized by CO2 narcosis and brains were processed to yield synaptosomes as previously described (Rothman et al., 2002). Synaptosomes were prepared from rat striatum for the DAT assays, whereas synaptosomes were prepared from whole brain minus striatum and cerebellum for the NET and SERT assays. For the release assays, 9 nM [3H]-MPP+ or 5 nM [3H]-dopamine was used to assess activity at DAT, 9 nM [3H]-MPP+ was used to assess activity at the NET, and 5 nM [3H]-5-HT was used to assess activity at SERT. All buffers used in the release assays contained 1 µM reserpine to block vesicular uptake of substrates. The selectivity of release assays was optimized for a single transporter by including unlabeled blockers to prevent uptake of substrates by competing transporters. Synaptosomes were preloaded with radiolabeled substrate in

Krebs-phosphate buffer (126 mM NaCl, 2.4 mM KCl, 0.5 mM KH2PO4, 1.1 mM CaCl2, 0.83 mM

MgCl2, 0.5 mM Na2SO4, 11.1 mM glucose, 13.7 mM Na2HPO4, 1 mg per mL ascorbic acid, and 0.05 mM pargyline, pH 7.4) to achieve steady state (DAT-1 h or 30 min, NET/SERT-1 h). Release assays were initiated by adding 850 µL of preloaded synaptosomes to 150 µL of test compound prepared in Krebs-phosphate buffer containing 1 mg mL-1 BSA. Maximal release was determined in the presence of (10 µM for DAT/NET and 100 µM for SERT). Assays were terminated (30 or 5 min for DAT, 30 min for NET, 5 min for SERT) by rapid vacuum filtration/washing through GF/B filters on a

0  Brandel harvester (Gaithersburg, MD, USA), and retained radioactivity was quantified by a PerkinElmer TopCount. Percent of maximal release was plotted against the log of compound concentration. Data were fit to a three-parameter logistic curve to generate EC50 values (GraphPad Prism 6.0, GraphPad Software, Inc., San Diego, CA, USA). Substrate activity was confirmed by detecting a significant reversal of the releasing effect of the test compound in the presence of selective uptake inhibitors (at least N=2).

2.5 Analysis and statistics All experimental data are represented as mean and S.E.M. For statistical comparison, FPM-triggered release of pre-loaded tritiated substrate via each individual transporter was analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison test. For FPM-triggered efflux in absence or presence of monensin, the data were analyzed with a Mann-Whitney test. P <0.05 was chosen as the minimum criterion for statistical significance.

1  3. Results and Discussion

The aim of the present study was to determine the molecular mechanism of action for the fluoro ring-substituted analogues 2-, 3-, and 4-FPM. Previous evidence shows the parent compound phenmetrazine is a substrate-type releaser at DAT and NET, with much weaker substrate activity at SERT (Rothman et al., 2002). Solis and colleagues recently reported that phenmetrazine induces an inwardly-directed sodium current in cells expressing DAT, consistent with its activity as a transportable DAT substrate (Solis et al., 2016). We speculated that addition of a fluorine substituent to the phenyl ring of phenmetrazine (Figure 1) might alter the relative potency of the isomers at SERT relative to DAT and NET. Data obtained here from uptake inhibition experiments in HEK293 cells show that the effects of FPM isomers are generally comparable to the parent compound phenmetrazine. More specifically, we found a pronounced selectivity for DAT and NET over SERT for 2-, 3- and 4-FPM. As depicted in Figure 2, each phenmetrazine was found to inhibit MAT- mediated uptake in a concentration-dependent manner. At DAT and NET, the test drugs potently 3 + inhibited the uptake of [ H]-MPP with IC50 values in the low micromolar range. Uptake at SERT was markedly less pronounced with corresponding IC50 values in the range of 40-300 µM (Figure 2 and Table 1). Notably, movement of the fluoro substitution from the 2-, to 3-, to 4-position produced a progressive stepwise increase in potency at SERT without affecting potency at DAT. As mentioned in the Introduction, psychostimulants can be subdivided into cocaine-like blockers and amphetamine-like releasers. Uptake inhibition assays can be used to identify drugs that interact with MATs, but these assays cannot distinguish non-transported uptake inhibitors from transported substrates (Baumann et al., 2012). Hence, superfusion studies were performed to delineate the possible impact of 2-, 3- and 4-FPM on transporter-mediated reverse transport. These experiments allow for monitoring the time-dependent release of preloaded [3H]-MPP+ via hDAT and hNET or [3H]-5HT via hSERT in the absence or presence of test drugs. HEK293 cells expressing the human isoforms of MATs were pre-loaded with radiolabeled substrate and baseline efflux was recorded for each transporter. Subsequently, the cells were challenged with 5 µM of 2-, 3- or 4-FPM to measure evoked efflux. At DAT and NET, the fluorinated phenmetrazines had similar effects, as each FPM significantly elevated the release of tritiated substrate versus untreated basal efflux at DAT and NET (Figure 3). At SERT, more divergent effects on release were detected with the isomers. The addition of 3- and 4-FPM induced moderate release of [3H]-5HT whereas 2-FPM had no detectable effect on SERT mediated efflux at the concentration tested (Figure 3). The latter finding is consistent with the uptake inhibition experiments in which 3- and 4-FPM exerted weak but measurable inhibitory effects on SERT mediated uptake, whereas no obvious effect could be observed for 2-FPM at concentrations below 30 µM (Figure 2). One limitation of superfusion studies is the possibility of misinterpreting the effects of drug- induced MAT inhibition as true transporter-mediated efflux. As described in previous publications

2  (Scholze et al., 2000), inhibition of MATs by blockers may unmask a basal leak of radiolabeled substrate, which could then be interpreted as efflux (i.e. “pseudoefflux”). In order to verify the nature of the efflux measured here, we used the co-application of the Na+/H+ ionophore monensin which disrupts the sodium gradient established across cell membranes (Mollenhauer et al., 1990). Since MATs strictly depend on the sodium gradient as a driving force (Kristensen et al., 2011), elevated sodium concentrations at the cytoplasmic side of the membrane will augment efflux triggered by substrates but not blockers (Bonisch, 1986, Sitte and Freissmuth, 2010). Therefore, we conducted efflux experiments in the presence or absence of monensin. At all MATs included in our study, the co- application of monensin (10 µM) and 2-, 3- or 4-FPM resulted in a significantly higher fractional release of tritiated substrate in comparison to that observed in the absence of monensin (Figure 4). The fact that efflux triggered by all tested FPMs was augmented several-fold in the presence of monensin substantiates the interpretation that 2-, 3- and 4-FPM indeed act as MAT substrates. Based on the results obtained from efflux studies performed in heterologous expression systems, we sought to test the phenmetrazine isomers in synaptosomal preparations from native rat brain tissue. Synaptosomes contain the plasmalemmal transporters and the neuronal components involved in neurotransmission (Gray and Whittaker, 1962). Here synaptosomes were preloaded with [3H]- dopamine or [3H]-MPP+ for measuring reverse transport via DAT and NET, respectively, or [3H]-5HT for measuring reverse transport SERT. Increasing concentrations of FPMs evoked release of tritiated substrate in a concentration dependent manner via all three MATs. 2-, 3- and 4-FPM exhibited approximately equal potency as releasers at DAT and NET, with EC50 values in the nanomolar range (Figure 5 and Table 2). In accordance with uptake inhibition assays performed in HEK293 cells, a shift to the right could be observed for the potencies of the isomers as releasers of [3H]-5HT at SERT. Importantly, ring-substitution at the 4-position produced an increase in releasing potency at SERT compared to the other isomers, such that 4-FPM was only 3-fold selective for DAT versus SERT (Figure 5 and Table 2). Thus, the release experiments in synaptosomes agree with uptake data from HEK cells which demonstrate that fluoro ring-substitution of phenmetrazine, specifically at the 4- position, can increase potency at SERT relative to DAT or NET. In this study, no in vivo experiments were performed, and extrapolation of in vitro findings to possible effects in animals or humans must be made with caution. For example, it was previously shown (Rothman et al., 2002) that systemic administration of phenmetrazine results in dose-related elevations of extracellular dopamine and serotonin in rat nucleus accumbens, despite the fact that in vitro findings indicate phenmetrazine is at least 10-fold DAT-selective compared to SERT. The present in vitro data show that fluorination at the 2- or 3-position of phenmetrazine did not have a major impact on monoamine transporter activity as compared to the parent compound phemetrazine. Thus, it is tempting to speculate that 3-FPM will have in vivo effects that are similar to phenmetrazine itself. Indeed, user reports found on Internet forums (e.g. erowid.org, drugs-forum or bluelight.org) indicate 3-FPM produces psychostimulant-like subjective effects in recreational human users.

3  Controlled clinical studies would be needed, however, to assess the clinical effects in more detail. In addition, no data for potential activity at receptors are available for the phenmetrazines examined in this study. For instance, a previous study conclusively demonstrated that the psychoactive properties of methcathinone analogs are attributable to their activities at monoamine transporters and not at receptors (Simmler et al., 2014). Future studies should examine the receptor activities for FPM isomers. The data described in this study predict that all three phenmetrazine isomers will exert similar effects at DAT and NET which could contribute to significant risk for abuse and addiction. In this study, we also included the 2- and 4-FPM isomers which have yet to appear as “research chemicals”. Given the fluid nature of the NPS phenomenon and the changes associated with “catch-up” legislation (Brandt et al., 2014), the appearance of 3-FPM analogues and its isomers cannot be fully excluded.

4. Conclusion

The “research chemical” 3-FPM, and its two positional isomers 2-FPM and 4-FPM, have been identified as substrate-type releasing agents at monoamine transporters. All of the fluoro isomers are more selective for DAT and NET as compared to SERT, indicating a high potential for abuse and addiction. Fluorination at the 2-position of the phenyl ring results in reduced potency at SERT but does not affect the potencies at DAT and NET, as compared to 3- and 4-FPM. This study has characterized the molecular mechanism of action of a current NPS and two positional isomers that could appear as replacements in response to banning of 3-FPM.

Conflict of interest statement HHS has received honoraria for lectures and consulting from AbbVie, Lundbeck, MSD, Ratiopharm, Roche, Sanofi-Aventis and Serumwerk Bernburg (past 5 years). All other authors declare no conflict of interest.

Acknowledgements The research was supported by Austrian Research Fund/FWF grants F3506 and W1232 (H.H.S.) and DA12970 (B.E.B.); F.P.M. is a recipient of a DOC-fellowship of the Austrian Academy of Sciences; Portions of the work were supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health, USA (J.S.P and M.H.B.).

+*  References:

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Web- References EMCDDA: http://www.emcdda.europa.eu/activities/action-on-new-drugs UNODC, EWA: https://www.unodc.org/LSS/Home/NPS

+-  Figure 1

F O O O NH F NH NH

F 2-FPM 3-FPM 4-FPM

Figure 1: Chemical structures of fluorophenmetrazine isomers 2-, 3- and 4-FPM

+.  Figure 2

     

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Figure 2: Effect of fluorinated phenmetrazine on monoamine transporter mediated uptake in HEK293 cells. HEK293 cells were incubated with 0.1 µM [3H]5HT (1min; SERT) or 0.02 µM [3H]-MPP+ (3min, DAT and NET) and increasing concentrations of fluorinated phenmetrazines. Non-specific uptake was determined in presence of 10 µM paroxetine (SERT) or 10 µM mazindole (DAT, NET). Data are presented as mean ± S.E.M. obtained from 3-4 experiments performed in triplicate.

+/  Figure 3

                   

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Figure 3: Effect of fluorinated phenmetrazines on [3H]-outflow from monoamine transporter expressing HEK293 cells. Cells were preloaded with [3H]-MPP+ (DAT, NET) or [3H]5HT (SERT) and superfused. Addition of 2-, 3- and 4-FPM (5 µM) is indicated by black bar and arrow. Data are represented as mean ± S.E.M. obtained from 3-4 experiments performed in triplicate. For statistical comparisons, the mean of the fractions from t=4 to 8min was compared to the mean of the basal fractions at t=-4 to 0min; Kruskal-Wallis test followed by Dunn’s multiple comparison test. *=P<0.05, n.s. not significant.

+0  Figure 4

                

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Figure 4: Monoamine transporter mediated reverse transport triggered by fluorinated phenmetrazines is augmented by monensin. FPM-triggered reverse transport (5 µM) of preloaded [3H]5HT or [3H]-MPP+ via SERT or DAT and NET expressing HEK293 cells, respectively, was monitored in presence (open symbols) or absence (closed symbols) of

+1  monensin (MON, 10 µM). Black bars and arrows indicate addition of substances. Data are mean and S.E.M. obtained from 3-4 experiments performed in triplicate. For statistical comparison, efflux in presence or absence of monensin at t=4, 6, 8 and 10 min was normalized to basal efflux (mean t=-4 to 0 min) and effects at each transporter were compared by the Mann-Whitney test (*=P<0.05).

+2  Figure 5

                     

                      

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Figure 5: Fluorinated phenmetrazines trigger reverse transport of [3H]-DA, [3H]-MPP+, and [3H]-5HT from rat brain synaptosomes. Rat brain synaptosomes were pre-loaded with tritiated monoamine substrates and challenged with increasing concentrations of 2-,3- and 4-FPM. Activity of FPMs at DAT, NET or SERT was determined in presence of specific inhibitors as outlined in the materials and methods section. Data are shown as mean ± S.E.M. obtained from 3 experiments performed in duplicate (DAT, SERT) or triplicate (NET).

+3  Table 1

IC (µM) 50 DAT NET SERT

2-FPM 1.335 ± 0.09 1.292 ± 0.03 294.7 ± 2.39

3-FPM 1.158 ± 0.29 1.515 ± 0.17 82.92 ± 17.12

4-FPM 2.137 ± 0.39 2.104 ± 0.28 39.02 ± 5.23

Table 1: Obtained IC50 values for FPM-mediated monoamine transporter uptake inhibition in HEK293 cells. Values are given as mean ± S.E.M. obtained from nonlinear curve fits as shown in Figure 2.

,*  Table 2

EC (nM) 50

DAT NET SERT

2-FPM 127 ± 13 58.2 ± 7.8 2620 ± 310

3-FPM 43.3 ± 3.5 29.6 ± 4.0 2548 ± 273

4-FPM 214 ± 16 84 ± 22 739 ± 170

Table 2: Obtained EC50 values for FPM-triggered release via monoamine transporters in rat brain synaptosomes. Values are given as mean ± S.E.M. obtained from nonlinear curve fits as shown in Figure 5.

,+  Discussion  General discussion

The thesis at hand deals with the pharmacological characterization of drugs that are suspected to exert their psychoactive effects via an interaction with MATs. The experimental findings are already discussed in detail in the embedded manuscripts. However, some aspects may go beyond the individual discussions included therein and as such will be dealt with here. Also, an overarching view shall be presented to highlight the connections between the studies.

In general, compounds that target MATs provide the opportunity to treat mental disorders, but also have the potential to be used recreationally (Kristensen et al., 2011). In this regard, it is worth pointing out that consumption of psychostimulants may be accompanied with a high risk for addiction (Taylor et al., 2013). Chronic abuse of addictive substances can trigger neurophysiological adaptations that eventually lead to , tolerance and craving (Cami and Farre, 2003) and may even manifest in stereotypical behaviors (Fasano et al., 2008). Consequently, drug abuse represents a potential threat for fiscal systems and public health (Baumann and Volkow, 2016) (United Nations Office on Drugs and Crime, 2016). The omnipresent use of psychoactive substances is a constant and persistent problem of society. At present, an increase in the abuse of NPS can be observed (Baumann, 2016), since NPS exploit legal loopholes (Rosenauer et al., 2013) and depict legal and easily available alternatives to regulated drugs of abuse (Meyer, 2016). Disturbingly, drug-related online forums contain detailed trip-reports and recommendations for both dosage and route of administration of NPS (Baumann, 2016, Meyer, 2016). The versatile phenomenon of NPS brings forth synthetic surrogates for cannabis, hallucinogens, psychostimulants and even (Brandt et al., 2014, Baumeister et al., 2015, Meyer, 2016). However, the NPS market is dominated by synthetic cathinones and phenethylamines, which share stimulant properties (Tyrkko et al., 2016). Adverse and deleterious effects associated with the abuse of NPS have been reported (Spiller et al., 2011, Wood et al., 2011, Prosser and Nelson, 2012, Loi et al., 2015). Together with the potential risk for addiction, these features inevitably highlight the need for profound insight into the mechanisms by which these novel drugs exert their effects.  LML The first part of this thesis aimed to identify potential bioactive effects of the phase I metabolites of mephedrone. Evidence has been reported that the overall effects of some compounds might be contingent upon their metabolites (Rothman et al., 2002, Green et al., 2003, Hofmaier et al., 2014). The results obtained from in vitro experiments clearly demonstrate that the phase I metabolites of mephedrone, i.e. nor-, 4-OH- and dihydro-mephedrone, act as substrates at MATs, and are fully capable of inducing transporter-mediated release. Interestingly, only one metabolite, nor-mephedrone, affected both the neurochemistry and behavior in vivo, upon intravenous administration. Marked effects could be observed on extracellular 5-HT (Mayer, 2016). However, the findings reported in this publication do not suffice to conclude if the metabolites relevantly contribute to the effects of mephedrone in vivo. Nevertheless, as discussed briefly in the embedded publication (Mayer et al., 2016), various cytochrome P450 (CYP) enzymes are present in brain, including CYP2D6 (Siegle et al., 2001, Miksys and Tyndale, 2002, Miksys et al., 2002, Miksys and Tyndale, 2013) which is the most relevant enzyme for the breakdown of mephedrone (Pedersen et al., 2013). Consequently, detailed analysis of the pharmacokinetics of mephedrone and its metabolites is required to determine if the metabolites are formed within the brain, thus, could contribute to the psychoactive effects. It is tempting to speculate that the effects of nor-mephedrone on 5-HT account for the MDMA-like effects that have been associated with mephedrone (Green et al., 2014). Another challenge on pharmacological grounds arises from the chiral centers of mephedrone and its metabolites. Mephedrone exists as two enantiomers, R- and S- mephedrone. At DAT, both enantiomers display comparable potencies as releasers, whereas S-mephedrone is much more potent at SERT (Gregg et al., 2015). Similar findings have been published regarding nor-mephedrone (Hutsell et al., 2016). As it is the case for mephedrone (Pedersen et al., 2013), CYP2D6 is also involved in the degradation of MDMA (Green et al., 2003). In 2015, a detailed study on the metabolism of MDMA in humans became available, containing detailed information on the pharmacokinetic differences of the metabolites in their enantiopure form (Steuer et al., 2015). Given the different potencies of the respective enantiomers of mephedrone and nor-mephedrone at SERT and that metabolism may favor the formation of one (Steuer et al., 2015), future studies shall unravel the effects of nor-, 4-OH and dihydro-mephedrone at MATs in their enantiopure form. In contrast to other NPS and established drugs of abuse (Simmler et al., 2014, Rickli et

 LMM al., 2015), no information is available on the affinities of the mephedrone metabolites towards monoamine receptors. Expertise in this regard would further refine our understanding of the action of mephedrone in vivo. In combination with enantioselective and time-resolved analysis of the abundance of the metabolites in plasma, urine and brain upon systemic mephedrone administration, these findings would allow for estimating the overall-contribution of the metabolites to the in vivo effects of mephedrone. A challenging, yet interesting, feature of the NPS market is that some drugs rapidly appear and disappear (Meyer, 2016), while others persist on the drugs markets (Baumann, 2016). Mephedrone was banned in most countries around 2011 (Green et al., 2014, Karila et al., 2015). However, based on the analysis of public urinals (Archer et al., 2013, Archer et al., 2014) wastewater from cities (Castiglioni et al., 2015, Styszko et al., 2016) and biological specimens (Salomone et al., 2016), mephedrone appears to be still used and has become an established psychostimulant on the market (Hockenhull et al., 2016) (United Nations Office on Drugs and Crime, 2016), which warrants further research on mephedrone.

At present, scientific literature on FPMs is scarce, if not completely absent. With respect to the fluorinated phenmetrazines, i.e. 2-, 3- and 4-FPM, a recent publication reported that 3-FPM has been identified in individuals seeking medical care (Backberg et al., 2016). In addition to reports from drug user forums, this piece of information further suggests that 3-FPM is used on a recreational basis. In the embedded manuscript, entitled “Pharmacological characterization of fluorinated phenmetrazine "research chemicals"“, it has been speculated that 2- and 4-FPM could appear on the drug markets. Recently, 4-FPM was discussed in an online drug- forum (see online reference). Both, the scientific publication and the online-evidence, strengthen the need for a pharmacological characterization of FPMs. Moreover, first insights into the metabolism of 3-FPM have been revealed (Mardal et al., 2016). Consequently, future studies could test the metabolites of 3-FPM for their potential bioactive properties at MATs or toxic effects.

Monensin augmented the MAT-mediated release induced by the tested phase 1 metabolites of mephedrone and 2-, 3- and 4-FPM. Nevertheless, it is worth mentioning that collapsing the sodium gradient may not only promote the reversal of

 LMN the transport cycle, but could also induce a specific conformation, allowing for channel-like efflux events (Kahlig et al., 2005) to occur. Testing this interesting hypothesis shall be subject of future investigations.

Both studies embedded in this thesis did not examine the effects of the compounds under scrutiny at VMATs. Baumann and colleagues reported that mephedrone and methylone did not deplete brain 5-HT levels to the same extent as MDMA (Baumann et al., 2012). Based on earlier findings, showing that methylone and methcathinone do not inhibit vesicular uptake of 5-HT as potently as methamphetamine or MDMA (Cozzi et al., 1999), a hypothesis was put forth that the low affinity towards VMAT2 accounts for the reduced depletion of brain 5-HT (Baumann et al., 2012). Evidence in support of this hypothesis is derived from the finding that the concentration-response curve of mephedrone, when compared to MDMA, is shifted towards the right in assays that measure uptake of [3H]DA by human VMAT2 (Pifl et al., 2015). Inhibition of VMAT2 and the concomitant rise in cytosolic monoamines could result in the formation of reactive oxygen species with neurotoxic effects (Hansen et al., 2002, Bogen et al., 2003, Mosharov et al., 2009). This speculation is fueled by a study from Caudle. et al., who reported that low VMAT2 expression correlates with neurodegeneration (Caudle et al., 2007). Stereoselective analysis of mephedrone and its phase I metabolites could strengthen the hypothesis, that low potencies at VMAT2 are responsible for the reduced neurotoxicity of mephedrone. Given the speculation that the metabolites would inhibit vesicular uptake in the same tier of potency as mephedrone. In case of FPMs, the parental compound phenmetrazine is inactive at VMAT2 (Partilla et al., 2006). However, it can not be assumed with certainty that the same holds true for 2-,3- or 4-FPM.

The reduced (mephedrone), or virtually absent (phenmetrazine) effect at VMAT2 may raises the question, from which pool monoamines are released in vivo. Various studies support the concept that amphetamine-like releasers are contingent upon vesicular pools of monoamines (Florin et al., 1995, Freyberg et al., 2016). However, in vivo experiments also showed that amphetamine-like drugs still induce efflux of monoamines in reserpine-treated animals (Butcher et al., 1988, Callaway et al., 1989, Adell et al., 1993). These findings nourish the idea that amphetamine-like drugs also trigger the release of a newly synthesized pool of neurotransmitters

 LMO (Sulzer, 2011). Interestingly, the dose-response effects of MDMA and mephedrone on extracellular DA and 5-HT in NAc were somewhat similar upon systemic administration in rats. Considering that the respective EC50 values of mephedrone and MDMA - to trigger reverse transport - via DAT and SERT are comparable, these findings support the idea of mephedrone and MDMA triggering the release of newly synthesized DA and 5-HT at the doses tested in this study (Baumann et al., 2012). However, it could also be speculated that the high brain penetration of mephedrone, as it easily crosses the BBB (Green et al., 2014), compensates for its weak effect at VMAT2 as compared to MDMA. Importantly, neurotoxicity of psychostimulants corresponds to their mode of action, i.e. the ability to penetrate into the cytosol. Abuse of amphetamine-like drugs, especially methamphetamine, appears to correlate with an increased risk of Parkinson’s disease (Callaghan et al., 2012, Pifl et al., 2015). In 2014, Baumann et al., reported a strong correlation between substance induced currents through SERT and neurotoxicity on serotonergic neurons (Baumann et al., 2014a). Interestingly, the substances that exerted neurotoxic effects in this study also revealed quite remarkable affinity towards VMAT2 (Partilla et al., 2006). Hence, it can be speculated that the cumulative effects of concentrative transport into the cytosol via plasmalemmal transporters and profound interaction with VMAT2 partially explains the deleterious effects of some psychostimulants on monoaminergic neurons. For example, methamphetamine, MDMA and AMPH are well-established substrates of DAT, NET and SERT, and interact with VMAT2 (Cozzi et al., 1999, Partilla et al., 2006). Consequently, the proposed role of VMAT2 in in drug-induced neurotoxicity warrants further research on VMAT2 in psychostimulant action. Insights into the activities at the vesicular and plasmalemmal transporters of NPS could serve as valuable indicators to predict neurotoxic effects.

Psychostimulant action is far from being understood completely, which is illustrated by the absence of an ultimate model for releaser-induced reverse transport via MATs and the enigmatic observation that various highly potent DAT-inhibitors fail to induce rewarding stimuli in humans (Heal et al., 2014, Sitte and Freissmuth, 2015). A potential player in releaser-induced efflux of monoamines is depicted by promiscuous and polyspecific monoamine transporters. As of 2016, it is generally assumed that amphetamine-like releasers exert their effects via DAT, NET and

 LMP SERT (Torres et al., 2003, Gether et al., 2006, Kristensen et al., 2011). However, converging evidence has been reported that low-affinity/high-capacity transporters are essentially involved in the clearance of extracellular monoamines, namely the organic cation transporters 1 to 3 (OCT1-3) and the plasmalemmal monoamine transporter (PMAT) (Daws, 2009). Unlike the sodium-driven SLC6-transporters, the transport direction of these transporters is determined by the electrochemical gradient of their substrates (Engel and Wang, 2005, Koepsell et al., 2007). At present, their exact role in psychostimulant action remains enigmatic, whereas experimental evidence suggests a role of OCT3 in psychostimulant action. For instance, this corticosterone-sensitive transporter, also known as uptake2 (Koepsell et al., 2007), is expressed in the nervous system, including catecholaminergic neurons (Vialou et al., 2008). Furthermore, OCT3 serves as compensatory 5-HT clearance system in mice deficient in SERT (Baganz et al., 2008) and inhibition of OCT3 enhances the effects of SSRIs (Horton et al., 2013). Strikingly, genetic polymorphisms in the gene encoding OCT3 have been associated with methamphetamine abuse (Aoyama et al., 2006). AMPH is more potent as a drug than would be expected from its binding affinity to DAT (Ritz et al., 1987, Sulzer, 2011). This observation could be attributed to its ability to induce release, rather than to block DAT (Sulzer, 2011). However, it could also be speculated that releasers, such as AMPH, take effect via additional targets, besides DAT, NET and SERT. This assumption is especially tempting with respect to the problem that the current treatments for psychostimulant addiction, that target MATs, are ineffective (Howell and Negus, 2014, Phillips et al., 2014).

Conclusion and future prospects  In summary, the mechanism of action of psychostimulants relies on a series of complex interactions that still need to be unraveled in detail. In addition to established drugs of abuse, the occurrence of NPS depicts a challenge for society, health care and science. Consequently, it is imperative to gain further insights into the molecular underpinnings, which underlie the rewarding, addictive and potentially neurotoxic effects of these drugs. The flexibility of the NPS market brings forth an overwhelming number of different substances. Each of which is equipped with a unique pharmacological fingerprint (Rosenauer et al., 2013). Merging the knowledge of each substance and its structure activity relationship allows for generating a better

 LMQ understanding of the molecular determinants of carrier-mediated transport fluxes in either direction. Eventually – and hopefully, this knowledge may result in the development of better treatments for addiction and imbalances in monoamine homeostasis.

 LMR References

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 LOR Appendix 

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Title: Neurotransmitter transporters: Logged in as: molecular function of important Felix Mayer drug targets Account #: Author: Ulrik Gether,Peter H. 3001077006 Andersen,Orla M. Larsson,Arne Schousboe Publication: Trends in Pharmacological Sciences Publisher: Elsevier Date: July 2006 Copyright © 2006 Elsevier Ltd. All rights reserved.

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License Number 4034890756223 License date Jan 23, 2017 Licensed Content Elsevier Publisher Licensed Content Trends in Pharmacological Sciences Publication Licensed Content Title Neurotransmitter transporters: molecular function of important drug targets Licensed Content Author Ulrik Gether,Peter H. Andersen,Orla M. Larsson,Arne Schousboe Licensed Content Date July 2006 Licensed Content Volume 27 Licensed Content Issue 7 Licensed Content Pages 9 Type of Use reuse in a thesis/dissertation Portion figures/tables/illustrations Number of 1 figures/tables/illustrations Format both print and electronic Are you the author of this No Elsevier article? Will you be translating? No Order reference number Original figure numbers Figure 1 Title of your Unravelling the mechanism of action of new psychoactive substances and their phase 1 thesis/dissertation metabolites Expected completion date Jan 2017 Estimated size (number 125 of pages) Elsevier VAT number GB 494 6272 12 Requestor Location Felix Mayer Waehringerstrasse 13A

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David R. Sibley January 24, 2017 President Bethesda, Maryland Felix Paul Mayer John D. Schuetz Institute of Pharmacology President-Elect St. Jude Children's Research Hospital Medical University of Vienna Währingerstrasse 13A Kenneth E. Thummel Past President Vienna 1090 University of Washington Austria Charles P. France Secretary/Treasurer Email: [email protected] The University of Texas Health Science Center at San Antonio Dear Felix Mayer: John J. Tesmer Secretary/Treasurer-Elect University of Michigan This is to grant you permission to reproduce the following figure in your thesis

Dennis C. Marshall titled “Unravelling the mechanism of action of new psychoactive substances and Past Secretary/Treasurer their phase 1 metabolites” for the Medical University of Vienna: Ferring Pharmaceuticals, Inc.

Margaret E. Gnegy Figure 8A from Anders S. Kristensen, Jacob Andersen, Trine N. Councilor University of Michigan Medical School Jørgensen, Lena Sørensen, Jacob Eriksen, Claus J. Loland, Kristian Strømgaard, and Ulrik Gether, SLC6 Neurotransmitter Transporters: Wayne L. Backes Structure, Function, and Regulation, Pharm Rev 2011, 63(3):585-640; Councilor Louisiana State University Health DOI: http://dx.doi.org/10.1124/pr.108.000869 Sciences Center

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Title: Amphetamines, new Logged in as: psychoactive drugs and the Felix Mayer monoamine transporter cycle Account #: Author: Harald H. Sitte,Michael 3001077006 Freissmuth Publication: Trends in Pharmacological Sciences Publisher: Elsevier Date: January 2015 Copyright © 2014 The Authors. Published by Elsevier Ltd.

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